Delta 4, 5 glycuronidase compositions and methods related thereto

ABSTRACT

The invention relates to Δ4,5 glycuronidase, related compositions, and methods of use thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/616,764, filed Nov. 11, 2009, now allowed, which is a continuation ofU.S. patent application Ser. No. 11/402,491, filed Apr. 11, 2006, nowU.S. Pat. No. 7,695,711, which is a divisional of U.S. patentapplication Ser. No. 10/429,921, filed May 5, 2003, now abandoned, whichclaims priority under 35 U.S.C. §119 from U.S. Provisional ApplicationNo. 60/377,488, filed May 3, 2002, the entire contents of each of whichare incorporated by reference herein.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. R01GM57073, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to Δ4, 5 glycuronidase and uses thereof. Inparticular, the invention relates to substantially pure Δ4, 5glycuronidase which is useful for a variety of purposes, includinganalysis of glycosaminoglycans (GAGs), sequencing, identifying,quantifying and purifying glycosaminoglycans present in a sample,removing glycosaminoglycans, such as heparin, from a solution andinhibiting angiogenesis, controlling coagulation, etc. The inventionalso relates to methods of treating cancer and inhibiting cellularproliferation and/or metastasis using Δ4, 5 glycuronidase and/or GAGfragments produced by enzymatic cleavage with Δ4, 5 glycuronidase.

BACKGROUND OF THE INVENTION

Glycosaminoglycans (GAGs) are linear, acidic polysaccharides that existubiquitously in nature as residents of the extracellular matrix and atthe cell surface of many different organisms of divergent phylogeny[Habuchi, O. (2000) Biochim Biophys Acta 1474, 115-27; Sasisekharan, R.,Bulmer, M., Moremen, K. W., Cooney, C. L., and Langer, R. (1993) ProcNatl Acad Sci USA 90, 3660-4]. In addition to a structural role, GAGsact as critical modulators of a number of biochemical signaling events[Tumova, S., Woods, A., and Couchman, J. R. (2000) Int J Biochem CellBiol 32, 269-88] requisite for cell growth and differentiation, celladhesion and migration, and tissue morphogenesis.

Heparan sulfate like glycosaminoglycans (GAGS or HSGAGs) are presentboth at the cell surface and in the extracellular matrix. Heparin-likeglycosaminoglycans are important components of the extracellular matrixthat are believed to regulate a wide variety of cellular activitiesincluding invasion, migration, proliferation and adhesion (Khodapkar, etal. 1998; Woods, et al., 1998). HSGAGs accomplish some of thesefunctions by binding to and regulating the biological activities ofdiverse molecules, including growth factors, morphogens, enzymes,extracellular proteins. HSGAGs are a group of complex polysaccharidesthat are variable in length, consisting of a disaccharide repeat unitcomposed of glucosamine and an uronic acid (either iduronic orglucuronic acid). The high degree of complexity for HSGAGs arises notonly from their polydispersity and the possibility of two differenturonic acid components, but also from differential modification at fourpositions of the disaccharide unit. Three positions, viz., C2 of theuronic acid and the C3, C6 positions of the glucosamine can beO-sulfated. In addition, C2 of the glucosamine can be N-acetylated orN-sulfated. Together, these modifications could theoretically lead to 32possible disaccharide units, making HSGAGs potentially more informationdense than either DNA (4 bases) or proteins (20 amino acids). It is thisenormity of possible structural variants that allows HSGAGs to beinvolved in a large number of diverse biological processes, includingangiogenesis (Sasisekharan, R., Moses, M. A., Nugent, M. A., Cooney, C.L. & Langer, R. (1994) Proc Nail Acad Sci U S A, 1524-8.), embryogenesis(Binari, R. et al (1997) Development, 2623-32; Tsuda, M., et al. (1999)Nature, 276-80.; and Lin, X., et al (1999) Development, 3715-23.) andthe formation of β-fibrils in Alzheimer's disease (McLaurin, J., et al(1999) Eur J Biochem, 1101-10. and Lindahl, B., et al (1999) J BiolChem, 30631-5).

One specific example of an HSGAG is heparin. Heparin, a highly sulphatedHSGAG produced by mast cells, is a widely used clinical anticoagulant,and is one of the first biopolymeric drugs and one of the fewcarbohydrate drugs. Heparin primarily elicits its effect through twomechanisms, both of which involve binding of antithrombin III (AT-III)to a specific pentasaccharide sequence,H_(NAc/S,6S)GH_(NS,3S,6S)I_(2S)H_(NS,6S) contained within the polymer.HSGAGs have also emerged as key players in a range of biologicalprocesses that range from angiogenesis [Folkman, J., Taylor, S., andSpillberg, C. (1983) Ciba Found Symp 100, 132-49] and cancer biology[Blackhall, F. H., Merry, C. L., Davies, E. J., and Jayson, G. C. (2001)Br J Cancer 85, 1094-8] to microbial pathogenesis [Shukla, et al (1999)Cell 99, 13-22]. HSGAGs have also recently been shown to play afundamental role in multiple aspects of development [Perrimon, N. andBernfield, M. (2000) Nature 404, 725-8]. The ability of HSGAGs toorchestrate multiple biological events is again likely a consequence ofits structural complexity and information density [Sasisekharan, R. andVenkataraman, G. (2000) Curr Opin Chem Biol 4, 626-31].

Although the structure and chemistry of HSGAGs are fairly wellunderstood, information on how specific HSGAG sequences modulatedifferent biological processes has proven harder to obtain.Determination of these HSGAG sequence has been technically challenging.HSGAGs are naturally present in very limited quantities, which, unlikeother biopolymers such as proteins and nucleic acids, cannot be readilyamplified. Second, due to their highly charged character and structuralheterogeneity, HSGAGs are not easily isolated from biological sources ina highly purified state. Additionally, the lack of sequence-specifictools to cleave HSGAGs in a manner analogous to DNA sequencing orrestriction mapping has made sequencing a challenge.

Recently, in an effort to develop an understanding of HSGAG structure,focus has been placed on the cloning and characterization of the enzymesinvolved in HSGAG biosynthesis. Another, strategy for elucidating thestructure of HSGAGs has been to employ specific HSGAG degradationprocedures, including chemical or enzymatic cleavage, in conjunctionwith analytical methodologies, including gel electrophoresis or HPLC, tosequence HSGAGs. Recently, we have introduced a sequencing procedurethat couples a bioinformatics framework with mass spectrometric andcapillary electrophoretic procedures to sequence rapidly biologicallyimportant HSGAGs, including saccharide sequences involved in modulatinganticoagulation. The sequencing methodology uses chemical and enzymatictools to modify or degrade an unknown glycosaminoglycan polymer in asequence-specific manner. (Venkataraman, G., et al., Science, 286,537-542 (1999), and U.S. patent applications Ser. Nos. 09/557,997 and09/558,137, both filed on Apr. 24, 2000, having common inventorship).

SUMMARY OF THE INVENTION

Δ4, 5 glycuronidase has been cloned from the F. heparinum genome and itssubsequent recombinant expression in E. coli as a soluble, highly activeenzyme has been accomplished. Thus, in one aspect the present inventionprovides for a substantially pure Δ4,5 glycuronidase. In one embodimentof the invention the substantially pure Δ4,5 glycuronidase is arecombinantly produced glycuronidase. Recombinant expression may beaccomplished in one embodiment with an expression vector. An expressionvector may be a nucleic acid for SEQ ID NO:2, optionally operably linkedto a promoter. In another embodiment the expression vector may be anucleic acid for SEQ ID NO:4 or a variant thereof also optionally linkedto a promoter. In one embodiment the substantially pure Δ4,5glycuronidase is produced using a host cell comprising the expressionvector. In another embodiment the substantially pure Δ4,5 glycuronidaseis a synthetic glycuronidase.

In another aspect the glycuronidase of the invention is a polypeptidehaving an amino acid sequence of SEQ ID NO:1, or a functional variantthereof. In yet another aspect the polypeptide has an amino acidsequence of SEQ ID NO:3, or a functional variant thereof.

In yet another aspect of the invention the polypeptide of the Δ4,5glycuronidase is an isolated polypeptide. The isolated polypeptide insome embodiments is set forth in SEQ ID NO:1 or is a functional variantthereof. In other embodiments the isolated polypeptide is set forth inSEQ ID NO:3 or a functional variant thereof.

In one aspect, the invention is a composition comprising, an isolatedΔ4,5 unsaturated glycuronidase having a higher specific activity thannative glycuronidase. In some embodiments, the specific activity is atleast about 60 picomoles of substrate hydrolyzed per minute per picomoleof enzyme. In one embodiment the Δ4,5 glycuronidase has a specificactivity that is about 2 fold higher than the native enzyme. In anotherembodiment the Δ4,5 glycuronidase has a specific activity that is about3 fold higher. The specific activity of the Δ4,5 glycuronidase in otherembodiments may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100 or any integer therebetween fold higher than the activity of thenative enzyme.

In yet another aspect of the invention an isolated nucleic acid moleculeis provided.

The nucleic acid is (a) nucleic acid molecules which hybridize understringent conditions to a nucleic acid molecule having a nucleotidesequence set forth as SEQ ID NO:2 or SEQ ID NO:4, and which code forΔ4,5 unsaturated glycuronidase having an amino acid sequence set forthas SEQ ID NO:1 or SEQ ID NO:3, respectively, (b) nucleic acid moleculesthat differ from the nucleic acid molecules of (a) in codon sequence dueto degeneracy of the genetic code, or (c) complements of (a) or (b). Inone embodiment the isolated nucleic acid molecule codes for SEQ ID NO:1.In another embodiment the isolated nucleic acid molecule comprises thenucleotide sequence set forth as SEQ ID NO:2. In still other embodimentsthe isolated nucleic acid molecule codes for SEQ ID NO:3 and in yetother embodiments the isolated nucleic acid molecule comprises thenucleotide sequence set forth as SEQ ID NO:4.

Pharmaceutical compositions of any of the compositions or vectorsdescribed herein are also encompassed in the invention.

In other aspects the invention relates to a method of cleaving aglycosaminoglycan with a Δ4,5 unsaturated glycuronidase. The method maybe performed by contacting a glycosaminoglycan with the glycuronidase inan effective amount to cleave the glycosaminoglycan. In one embodimentthe invention is a glycosaminoglycan prepared according to this method.

In other aspects the invention also provides a method of cleaving aglycosaminoglycan comprised of at least one disaccharide unit. Themethod may be performed by contacting the glycosaminoglycan with aglycuronidase of the invention in an effective amount to cleave theglycosaminoglycan. In some embodiments the glycosaminoglycan is a longchain saccharide. In other embodiments the glycosaminoglycan does notcontain a 2-0 sulfated uronidate or it does not contain N-substitutedglycosamine. In yet another embodiment the glycosaminoglycan is 6-0sulfated. The disaccharide units in some embodiments are ΔUH_(NAc);ΔUH_(NAc,6S; ΔUH) _(NS,6S); or ΔUH_(NS). In another embodiment theinvention also provides for the products of the cleavage of aglycosaminoglycan with the Δ4,5 glycuronidase. In some embodiments theglycuronidase is used to generate a LMWH.

The present invention also provides methods for the analysis ofglycosaminoglycan. In one aspect the invention is a method of analyzinga glycosaminoglycan by contacting a glycosaminoglycan with theglycuronidase of the invention in an effective amount to analyze theglycosaminoglycan. In one embodiment the method is a method foridentifying the presence of a particular glycosaminoglycan in a sample.In another embodiment the method is a method for determining theidentity of a glycosaminoglycan in a sample. In yet another embodimentthe method is a method for determining the purity of a glycosaminoglycanin a sample. In still a further embodiment the method is a method fordetermining the composition of a glycosaminoglycan in a sample. Inanother embodiment the method is a method for determining the sequenceof saccharide units in a glycosaminoglycan. In other embodiments, thesemethods may also comprise an additional analytical technique such asmass spectrometry, gel electrophoresis, capillary electrophoresis andHPLC. In some embodiments the glycosaminoglycan is LMWH.

In other aspects the invention is a method of removing heparin from aheparin containing fluid by contacting a heparin containing fluid with aglycuronidase of the invention in an effective amount to remove heparinfrom the heparin containing fluid. In one embodiment the glycuronidaseis immobilized on a solid support. In another embodiment a heparinase isalso provided and the heparinase is also immobilized on the solidsupport.

In another aspect the invention is a method of inhibiting angiogenesisby administering to a subject in need thereof an effective amount of anyof the pharmaceutical preparations described herein for inhibitingangiogenesis.

In another aspect a method of treating cancer by administering to asubject in need thereof an effective amount of any of the pharmaceuticalpreparations described herein for treating cancer is also provided.

Yet another aspect of the invention is a method of inhibiting cellularproliferation by administering to a subject in need thereof an effectiveamount of any of the pharmaceutical preparations described herein forinhibiting cellular proliferation.

In another aspect a method of treating a coagulation disease byadministering to a subject in need thereof a LMWH prepared using theglycuronidase of the invention.

In some embodiments of the methods of the invention the glycuronidase isused concurrently with or following treatment with heparinase.

In other aspects of the invention, the pharmaceutical compositions andtherapeutic methods are provided using the Δ4,5 unsaturatedglycuronidase and the cleaved GAG fragments alone or in combination.

Other aspects of the invention provide compositions that include otherenzymes such as heparinase with the Δ4,5 unsaturated glycuronidase.

In other aspects a pharmaceutical preparation of a composition or vectorof the invention in a pharmaceutically acceptable carrier is provided.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the purification of Δ4, 5 glycuronidase fromFlavobacterium and resultant proteolysis. A. Gel filtrationchromatography of the purified enzyme. B. Purification of Δ4, 5 peptidesby reverse phase HPLC following trypsinization of the native protein. C.Amino acid sequence of select peptides isolated in B. Peaks 8, 12, 13,19, 24 and 26 are SEQ ID NOs: 18-23, respectively.

FIG. 2 provides a schematic map of A4, 5 genomic clones. A. Partialcarboxy-terminal clones G5A and G5H (black arrows) were isolated byhybridization screening of a λZAP Flavobacterial library using probes 1and 2, respectively. Also shown is the Eco R1 restriction sitedelimiting the 5′ end of GSA. B. Strategy to obtain the Δ4, 5 5′terminus by Southern hybridization. Shown are the autoradiogram and itscorresponding restriction map. Genomic DNA was restricted with Eco R1alone (lane 1) or as a double digest with Hind III (lane 2), Barn H1(lane3), or Bgl II (lane 4), respectively. DNA hybridization probe 3used was amplified by PCR using N-terminal primers 68 and 74, both ofwhich are 5′ to the Eco R1 site. The Bgl II-Eco R1˜1.5 kb DNA fragment(gray bar) was isolated for subcloning and DNA sequencing. C. Schematicrepresentation of the full-length Δ4, 5 gene (1.2 kb) compiled fromoverlapping clones shown in A. and B.

FIG. 3 depicts the Δ4, 5 glycuronidase gene sequence. Full-length genewas isolated using methods outlined in FIG. 2. The amino acid andnucleic acid sequences are given in SEQ ID NOS: 3 and 4, respectively.Shown here are both the coding and flanking DNA sequences. The CDS(coding sequence) of 1209 base pairs contains an ORF encoding a putativeprotein of 402 amino acids. Initiation and termination codons arehighlighted in bold. A possible Shine-Dalgarno (SD) sequence is boxed.The presumed signal sequence is underlined and its cleavage sitedelimited by a vertical arrow. The Eco R1 restriction site isdouble-overscored. Also shown are the degenerate primer pairs (shown asarrows) used to PCR amplify DNA hybridization probes 1 and 2 as well asthe relative positions of purified Δ4, 5 peptides (shaded in gray) forwhich direct sequence information was obtained.

FIG. 4 illustrates the Δ4, 5 glycuronidase primary sequence analyses. A.Hydropathy plot (Kyte-Doolittle). Positive values represent increasinghydrophobicity. B. Theoretical signal sequence determination using aminoacids 1-65. Indices were calculated using SignalP V.1.1 using networkstrained on gram-negative bacteria. Putative cleavage site locatedbetween G20 and M21 is represented by a vertical arrow. C. CLUSTAL Wmultiple alignment of full-length Δ4, 5 glycuronidase with selectglucuronyl hydrolases. Protein sequences were selected from an initialBLASTP search of the protein database. Identical amino acids are shadedin dark gray, near invariant positions in charcoal, and conservativesubstitutions in light gray. Gen Bank accession numbers are as follows:Bacillus sp. (AB019619); Streptococcus pneumoniae (AE008410);Streptococcus pyogenes (AE006517); Agaricus bisporsus (AJ271692);Bactobacillus halodurans (AP001514).

FIG. 5 provides results of recombinant Δ4,5^(Δ°) protein expression andpurification. The amino acid and nucleic acid sequences are given as SEQID NOS: 1 and 2, respectively. SDS-PAGE of A4, 5 protein fractions atvarious purification stages following expression in BL21 (DE3) as a6XHIS N-terminal fusion protein. Shown here is a 12% gel that is stainedwith Coomassie-Brilliant blue. Lane 2, lysate from uninduced bacterialcells; Lane 3, crude cell lysate from induced cultures; Lane 4, Ni⁺²chelation chromatography purification; Lane 5, thrombin cleavage toremove N-terminal 6× His purification tag. Molecular weight markers(Lanes 1 and 6) are also noted.

FIG. 6 depicts the effects of Δ4, 5 glycuronidase biochemical reactionconditions. A. [NaCl] titration; B. Effect of reaction temperature C. pHprofile. Relative enzyme activities were derived from the initial ratesnormalized to 100 mM NaCl (A) or 30° C. (C). k_(cat) and K_(m) valuesfor the pH profile were extrapolated from Michaelis-Menten kinetics asdescribed in the Methods (and FIG. 8) The disulfated heparindisaccharide ΔUH_(NS,6S) was used in all three experiments.

FIG. 7 depicts a kinetic comparison of native and recombinant enzymes.Relative specific activities were measured for both enzyme fractionsunder identical reaction conditions that included 200 nM enzyme and 500μM of the heparin disaccharide substrate (ΔUHNAc). Flavobacterial Δ4, 5(closed circles); recombinant Δ4, 5 (open circles).

FIG. 8 illustrates disaccharide substrate specificity. A. Kineticprofiles for heparin disaccharides of varying sulfation. Initial rateswere determined using 200 nM enzyme under standard conditions. Vo vs.[S] curves were fit to Michaelis-Menten steady state kinetics using anon-linear least squares analysis. B. Lineweaver-Burke representation ofthe data shown in A. ΔUH_(Nac,6S) (); ΔUH_(Nac) (O); ΔUH_(NS,6S) (▴);ΔH_(NS) (Δ); ΔUH_(NH2,16S) (+) ΔU_(2S)H_(NS)(+, no activity).

FIG. 9 depicts the tandem use of heparinases and Δ4, 5 glycuronidase inHSGAG compositional analyses. 200 m heparin was exhaustively digestedwith heparinases I, II, and III, after which Δ4, 5 was added for avarying length of time. disaccharide products were resolved by capillaryelectrophoresis. Assignment of saccharide composition shown for eachpeak was confirmed by MALDI-MS. A., minus Δ4, 5 enzyme control; dashedline, B., minute (partial) Δ4, 5 incubation; C., 30 minute (exhaustive)Δ4, 5 incubation.

DETAILED DESCRIPTION OF THE INVENTION

The invention in some aspects relates to Δ4, 5 glycuronidase,substantially pure forms thereof and uses thereof. In particular theinvention arose, in part, from the cloning of Δ4, 5 glycuronidase thatnow enables one of skill in the art to produce the enzyme in largequantities and in substantially pure form. The invention also providesanother tool that may be used to determine the structure ofglycosaminoglycans and to help elucidate their role in cellularprocesses. It has now also been discovered that substantially purepreparations of

Δ4, 5 glycuronidase having higher specific activity than the enzymeproduced from culture may be produced. The invention also provides forcleavage of glycosaminoglycans (GAGs) as well as for the analysis of asample of GAGs and for their sequencing. This present invention alsoprovides treatment and prevention methods for cancer through the controlof cellular proliferation, angiogenesis and/or coagulation disorderswith the enzyme and/or its cleavage products (GAG fragments).

One aspect of the invention enables one of ordinary skill in the art, inlight of the present disclosure, to produce substantially purepreparations of the Δ4, 5 glycuronidase by standard technology,including recombinant technology, direct synthesis, mutagenesis, etc.For instance, using recombinant technology one may produce substantiallypure preparations of the Δ4, 5 glycuronidase having the amino acidsequences of SEQ ID NO:1 or encoded by the nucleic acid sequence of SEQID NO:2. In other aspects of the invention substantially purepreparations of the Δ4, 5 glycuronidase having the amino acid sequencesof SEQ ID NO:3 or encoded by the nucleic acid sequence of SEQ ID NO:4can be prepared. One of skill in the art may also substitute appropriatecodons to produce the desired amino acid substitutions in SEQ ID NOs:1or 3 by standard site-directed mutagenesis techniques. One may also useany sequence which differs from the nucleic acid equivalents of SEQ IDNO:1 or 3 only due to the degeneracy of the genetic code as the startingpoint for site directed mutagenesis. The mutated nucleic acid sequencemay then be ligated into an appropriate expression vector and expressedin a host such as E. coli. The resultant Δ4, 5 glycuronidase may then bepurified by techniques, including those disclosed below.

As used herein, the term “substantially pure” means that the proteinsare essentially free of other substances to an extent practical andappropriate for their intended use. In particular, the proteins aresufficiently pure and are sufficiently free from other biologicalconstituents of their hosts cells so as to be useful in, for example,protein sequencing, or producing pharmaceutical preparations.

As used herein, a “substantially pure Δ4,5 unsaturated glycuronidase” isa preparation of Δ4,5 unsaturated glycuronidase which has been isolatedor synthesized and which is greater than about 90% free of contaminants.A contaminant is a substance with which the Δ4,5 unsaturatedglycuronidase is ordinarily associated in nature that interfere with theactivity of the enzyme. Preferably, the material is greater than about91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even greater than about 99%free of contaminants. The degree of purity may be assessed by meansknown in the art. One method for assessing the purity of the materialmay be accomplished through the use of specific activity assays. Thenative Δ4,5 glycuronidase which has been described in the prior art asbeing isolated from F. heparinum has low specific activity because ofimpurities inherent in harvesting the enzyme from bacterial cultures ofF. heparinum.

The invention also provides isolated polypeptides (including wholeproteins and partial proteins), of Δ4, 5 glycuronidase having the aminoacid sequence of SEQ ID NO:1 and functional variants thereof. Isolatedpolypeptides are also provided by the invention that have the amino acidsequence of SEQ ID NO:3. Polypeptides can be isolated from biologicalsamples, and can also be expressed recombinantly in a variety ofprokaryotic and eukaryotic expression systems by constructing anexpression vector appropriate to the expression system, introducing theexpression vector into the expression system, and isolating therecombinantly expressed protein. Polypeptides can also be synthesizedchemically using well-established methods of peptide synthesis.

As used herein with respect to polypeptides, “isolated” means separatedfrom its native environment and present in sufficient quantity to permitits identification or use.

Isolated, when referring to a protein or polypeptide, means, forexample: (i) selectively produced by expression cloning or (ii) purifiedas by chromatography or electrophoresis. Isolated proteins orpolypeptides may be, but need not be, substantially pure. Because anisolated polypeptide may be admixed with a pharmaceutically acceptablecarrier in a pharmaceutical preparation, the polypeptide may compriseonly a small percentage by weight of the preparation. The polypeptide isnonetheless isolated in that it has been separated from the substanceswith which it may be associated in living systems, i.e., isolated fromother proteins.

Thus the term “Δ4, 5 glycuronidase polypeptides” embraces variants aswell as the natural Δ4, 5 glycuronidase polypeptides. As used herein, a“variant” of a Δ4, 5 glycuronidase polypeptide is a polypeptide whichcontains one or more modifications to the primary amino acid sequence ofa naturally occurring Δ4, 5 glycuronidase polypeptide. Variants includemodified Δ4, 5 glycuronidase polypeptides that do not have alteredfunction relative to the polypeptide of the unmodified (naturallyoccurring) sequence. Variants also include Δ4, 5 glycuronidasepolypeptides with altered function. Modifications which create a Δ4, 5glycuronidase polypeptide variant are typically made to the nucleic acidwhich encodes the Δ4, 5 glycuronidase polypeptide, and can includedeletions, point mutations, truncations, amino acid substitutions andaddition of amino acids or non-amino acid moieties to: 1) enhance aproperty of a Δ4, 5 glycuronidase polypeptide, such as protein stabilityin an expression system or the stability of protein-protein binding; 2)provide a novel activity or property to a Δ4, 5 glycuronidasepolypeptide, such as addition of a detectable moiety; or 3) to provideequivalent or better interaction with other molecules (e.g., heparin).Alternatively, modifications can be made directly to the polypeptide,such as by cleavage, addition of a linker molecule, addition of adetectable moiety, such as biotin, addition of a fatty acid, and thelike. Modifications also embrace fusion proteins comprising all or partof the Δ4, 5 glycuronidase amino acid sequence. One of skill in the artwill be familiar with methods for predicting the effect on proteinconformation of a change in protein sequence, and can thus “design” avariant Δ4, 5 glycuronidase polypeptide according to known methods. Oneexample of such a method is described by Dahiyat and Mayo in Science278:82-87, 1997, whereby proteins can be designed de novo. The methodcan be applied to a known protein to vary a only a portion of thepolypeptide sequence. By applying the computational methods of Dahiyatand Mayo, specific variants of a polypeptide can be proposed and testedto determine whether the variant retains a desired conformation.

Variants can include Δ4, 5 glycuronidase polypeptides which are modifiedspecifically to alter a feature of the polypeptide unrelated to itsphysiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of aΔ4, 5 glycuronidase polypeptide by eliminating proteolysis by proteasesin an expression system (e.g., dibasic amino acid residues in yeastexpression systems in which KEX2 protease activity is present).Mutations of a nucleic acid which encodes a Δ4, 5 glycuronidasepolypeptide preferably preserve the amino acid reading frame of thecoding sequence, and preferably do not create regions in the nucleicacid which are likely to hybridize to form secondary structures, such ahairpins or loops, which can be deleterious to expression of the variantpolypeptide. Mutations can be made by selecting an amino acidsubstitution, or by random mutagenesis of a selected site in a nucleicacid which encodes the polypeptide. Variant polypeptides are thenexpressed and tested for one or more activities to determine whichmutation provides a variant polypeptide with the desired properties.Further mutations can be made to variants (or to non-variant Δ4, 5glycuronidase polypeptides) which are silent as to the amino acidsequence of the polypeptide, but which provide preferred codons fortranslation in a particular host. The preferred codons for translationof a nucleic acid in, e.g., E. coli, are well known to those of ordinaryskill in the art. Still other mutations can be made to the noncodingsequences of a Δ4, 5 glycuronidase gene or cDNA clone to enhanceexpression of the polypeptide. One type of amino acid substitution isreferred to as a “conservative substitution.”

As used herein, a “conservative amino acid substitution” or“conservative substitution” refers to an amino acid substitution inwhich the substituted amino acid residue is of similar charge as thereplaced residue and is of similar or smaller size than the replacedresidue. Conservative substitutions of amino acids include substitutionsmade amongst amino acids within the following groups: (a) the smallnon-polar amino acids, A, M, I, L, and V; (b) the small polar aminoacids, G, S, T and C; (c) the amido amino acids, Q and N; (d) thearomatic amino acids, F, Y and W; (e) the basic amino acids, K, R and H;and (f) the acidic amino acids, E and D. Substitutions which are chargeneutral and which replace a residue with a smaller residue may also beconsidered “conservative substitutions” even if the residues are indifferent groups (e.g., replacement of phenylalanine with the smallerisoleucine). The term “conservative amino acid substitution” also refersto the use of amino acid analogs or variants.

Methods for making amino acid substitutions, additions or deletions arewell known in the art. The terms “conservative substitution”,“non-conservative substitutions”, “non-polar amino acids”, “polar aminoacids”, and “acidic amino acids” are all used consistently with theprior art terminology. Each of these terms is well-known in the art andhas been extensively described in numerous publications, includingstandard biochemistry text books, such as “Biochemistry” by GeoffreyZubay, Addison-Wesley Publishing Co., 1986 edition, which describesconservative and non-conservative substitutions, and properties of aminoacids which lead to their definition as polar, non-polar or acidic.

One skilled in the art will be able to predict the effect of asubstitution by using routine screening assays, preferably thebiological assays described herein. Modifications of peptide propertiesincluding thermal stability, enzymatic activity, hydrophobicity,susceptibility to proteolytic degradation or the tendency to aggregatewith carriers or into multimers are assayed by methods well known to theordinarily skilled artisan. For additional detailed description ofprotein chemistry and structure, see Schulz, G. E. et al., Principles ofProtein Structure, Springer-Verlag, New York, 1979, and Creighton, T.E., Proteins: Structure and Molecular Principles, W. H. Freeman & Co.,San Francisco, 1984.

Additionally, some of the amino acid substitutions are non-conservativesubstitutions. In certain embodiments where the substitution is remotefrom the active or binding sites, the non-conservative substitutions areeasily tolerated provided that they preserve a tertiary structurecharacteristic of, or similar to, native Δ4, 5 glycuronidase, therebypreserving the active and binding sites. Non-conservative substitutions,such as between, rather than within, the above groups (or two otheramino acid groups not shown above), which will differ more significantlyin their effect on maintaining (a) the structure of the peptide backbonein the area of the substitution (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

In another set of embodiments an isolated nucleic acid equivalent of SEQID NO:2 encode the substantially pure Δ4, 5 glycuronidase of theinvention and functional variants thereof. In still further embodimentsisolated nucleic acid equivalents of SEQ ID NO:4 are also given.According to the invention, isolated nucleic acid molecules that codefor a Δ4, 5 glycuronidase polypeptide are provided and include: (a)nucleic acid molecules which hybridize under stringent conditions to amolecule selected from a group consisting of the nucleic acid equivalentof SEQ ID NO:2 or 4 and which code for a Δ4, 5 glycuronidase polypeptideor parts thereof, (b) deletions, additions and substitutions of (a)which code for a respective Δ4, 5 glycuronidase polypeptide or partsthereof, (c) nucleic acid molecules that differ from the nucleic acidmolecules of (a) or (b) in codon sequence due to the degeneracy of thegenetic code, and (d) complements of (a), (b) or (c). The invention alsoincludes degenerate nucleic acids which include alternative codons tothose present in the naturally occurring materials. For example, serineresidues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Eachof the six codons is equivalent for the purposes of encoding a serineresidue. Thus, it will be apparent to one of ordinary skill in the artthat any of the serine-encoding nucleotide triplets may be employed todirect the protein synthesis apparatus, in vitro or in vivo, toincorporate a serine residue into an elongating Δ4, 5 glycuronidasepolypeptide. Similarly, nucleotide sequence triplets which encode otheramino acid residues include, but are not limited to: CCA, CCC, CCG andCCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons);ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparaginecodons); and ATA, ATC and ATT (isoleucine codons). Other amino acidresidues may be encoded similarly by multiple nucleotide sequences.Thus, the invention embraces degenerate nucleic acids that differ fromthe biologically isolated nucleic acids in codon sequence due to thedegeneracy of the genetic code.

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its naturally occurring state in its natural host is not. Anisolated nucleic acid may be substantially purified, but need not be.For example, a nucleic acid that is isolated within a cloning orexpression vector is not pure in that it may comprise only a tinypercentage of the material in the cell in which it resides. Such anucleic acid is isolated, however, as the term is used herein because itis readily manipulable by standard techniques known to those of ordinaryskill in the art. One embodiment of the invention provides Δ4, 5glycuronidase that is recombinantly produced. Such molecules may berecombinantly produced using a vector including a coding sequenceoperably joined to one or more regulatory sequences. As used herein, acoding sequence and regulatory sequences are said to be “operablyjoined” when they are covalently linked in such a way as to place theexpression or transcription of the coding sequence under the influenceor control of the regulatory sequences. If it is desired that the codingsequences be translated into a functional protein the coding sequencesare operably joined to regulatory sequences. Two DNA sequences are saidto be operably joined if induction of a promoter in the 5′ regulatorysequences results in the transcription of the coding sequence and if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region to direct the transcription of the codingsequences, or (3) interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a promoter regionwould be operably joined to a coding sequence if the promoter regionwere capable of effecting transcription of that DNA sequence such thatthe resulting transcript might be translated into the desired protein orpolypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Promoters may beconstitutive or inducible. Regulatory sequences may also includeenhancer sequences or upstream activator sequences, as desired.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of

DNA although RNA vectors are also available. Vectors include, but arenot limited to, plasmids and phagemids. A cloning vector is one which isable to replicate in a host cell, and which is further characterized byone or more endonuclease restriction sites at which the vector may becut in a determinable fashion and into which a desired DNA sequence maybe ligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium, or just a single time per host as thehost reproduces by mitosis. In the case of phage, replication may occuractively during a lytic phase or passively during a lysogenic phase. Anexpression vector is one into which a desired DNA sequence may beinserted by restriction and ligation such that it is operably joined toregulatory sequences and may be expressed as an RNA transcript. Vectorsmay further contain one or more marker sequences suitable for use in theidentification of cells which have or have not been transformed ortransfected with the vector. Markers include, for example, genesencoding proteins which increase or decrease either resistance orsensitivity to antibiotics or other compounds, genes which encodeenzymes whose activities are detectable by standard assays known in theart (e.g., 13-galactosidase or alkaline phosphatase), and genes whichvisibly affect the phenotype of transformed or transfected cells, hosts,colonies or plaques. Preferred vectors are those capable of autonomousreplication and expression of the structural gene products present inthe DNA segments to which they are operably joined.

As used herein, the term “stringent conditions” refers to parametersknown to those skilled in the art. One example of stringent conditionsis hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin (BSA),25 mM NaH₂PO₄ (pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodiumchloride/0.015 M sodium citrate, pH7; SDS is sodium dodecyisuiphate; andEDTA is ethylene diamine tetra acetic acid. There are other conditions,reagents, and so forth which can be used, which result in the samedegree of stringency. A skilled artisan will be familiar with suchconditions, and thus they are not given here.

The skilled artisan also is familiar with the methodology for screeningcells for expression of such molecules, which then are routinelyisolated, followed by isolation of the pertinent nucleic acid. Thus,homologs and alleles of the substantially pure Δ4, 5 glycuronidase ofthe invention, as well as nucleic acids encoding the same, may beobtained routinely, and the invention is not intended to be limited tothe specific sequences disclosed. It will be understood that the skilledartisan will be able to manipulate the conditions in a manner to permitthe clear identification of homologs and alleles of the Δ4, 5glycuronidase nucleic acids of the invention. The skilled artisan alsois familiar with the methodology for screening cells and libraries forexpression of such molecules which then are routinely isolated, followedby isolation of the pertinent nucleic acid molecule and sequencing.

In general homologs and alleles typically will share at least about 40%nucleotide identity and/or at least about 50% amino acid identity withthe equivalents of SEQ ID Nos: 2 and 1, respectively. Homologs andalleles of the invention are also intended to encompass the nucleic acidand amino acid equivalents of SEQ ID Nos: 4 and 3, respectively. In someinstances sequences will share at least about 50% nucleotide identityand/or at least about 65% amino acid identity and in still otherinstances sequences will share at least about 60% nucleotide identityand/or at least about 75% amino acid identity. The homology can becalculated using various, publicly available software tools developed byNCBI (Bethesda, Md.) that can be obtained through the internet(ftp:/ncbi.nlm.nih.gov/pub/). Exemplary tools include the BLAST systemavailable at http://wwww.ncbi.nlm.nih.gov. Pairwise and ClustalWalignments (BLOSUM30 matrix setting) as well as Kyte-Doolittlehydropathic analysis can be obtained using the MacVetor sequenceanalysis software (Oxford Molecular Group). Watson-Crick complements ofthe foregoing nucleic acids also are embraced by the invention.

In screening for Δ4, 5 glycuronidase related genes, such as homologs andalleles of Δ4, 5 glycuronidase, a Southern blot may be performed usingthe foregoing conditions, together with a radioactive probe. Afterwashing the membrane to which the DNA is finally transferred, themembrane can be placed against X-ray film or a phosphoimager plate todetect the radioactive signal.

For prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors include pBR322, pUC18, pUC 19 and the like; suitable phage or bacteriophage vectors includeλgt10, λgt11 and the like; and suitable virus vectors include pMAM-neo,pKRC and the like. Preferably, the selected vector of the presentinvention has the capacity to autonomously replicate in the selectedhost cell. Useful prokaryotic hosts include bacteria such as E. coli,Flavobacterium heparinum, Bacillus, Streptomyces, Pseudomonas,Salmonella, Serratia, and the like.

To express the substantially pure Δ4, 5 glycuronidase of the inventionin a prokaryotic cell, it is desirable to operably join the nucleic acidsequence of a substantially pure Δ4, 5 glycuronidase of the invention toa functional prokaryotic promoter. Such promoter may be eitherconstitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, the bla promoter of the β-lactamase genesequence of pBR322, and the CAT promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, and the like. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage λ (P_(L) and P_(R)), the trp, recA, lacZ, lacI, and galpromoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol.162:176-182 (1985)) and the ζ-28-specific promoters of B. subtilis(Gilman et al., Gene sequence 32:11-20 (1984)), the promoters of thebacteriophages of Bacillus (Gryczan, In: The Molecular Biology of theBacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters(Ward et al., Mol. Gen. Genet. 203:468-478 (1986)).

Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol.1:277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986)); andGottesman (Ann. Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the encoding sequence. Such ribosomebinding sites are disclosed, for example, by Gold et al. (Ann. Rev.Microbiol. 35:365-404 (1981)).

Because prokaryotic cells may not produce the Δ4, 5 glycuronidase of theinvention with normal eukaryotic glycosylation, expression of the Δ4, 5glycuronidase of the invention of the eukaryotic hosts is useful whenglycosylation is desired. Preferred eukaryotic hosts include, forexample, yeast, fungi, insect cells, and mammalian cells, either in vivoor in tissue culture. Mammalian cells which may be useful as hostsinclude HeLa cells, cells of fibroblast origin such as VERO or CHO-K1,or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or themyeloma P3x63Sg8, and their derivatives. Preferred mammalian host cellsinclude SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR332 that may provide better capacities for correct post-translationalprocessing.

Embryonic cells and mature cells of a transplantable organ also areuseful according to some aspects of the invention.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.

Another preferred host is an insect cell, for example in Drosophilalarvae. Using insect cells as hosts, the Drosophila alcoholdehydrogenase promoter can be used (Rubin, Science 240:1453-1459(1988)). Alternatively, baculovirus vectors can be engineered to expresslarge amounts of the Δ4, 5 glycuronidase of the invention in insectcells (Jasny, Science 238:1653 (1987); Miller et al., In: GeneticEngineering (1986), Setlow, J. K., et al., eds., Plenum, Vol. 8, pp.277-297).

Any of a series of yeast gene sequence expression systems whichincorporate promoter and termination elements from the genes coding forglycolytic enzymes and which are produced in large quantities when theyeast are grown in media rich in glucose may also be utilized. Knownglycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provide substantial advantages inthat they can also carry out post-translational peptide modifications. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number plasmids which can be utilized forproduction of the desired proteins in yeast. Yeast recognize leadersequences on cloned mammalian gene sequence products and secretepeptides bearing leader sequences (i.e., pre-peptides).

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus, simian virus,or the like, where the regulatory signals are associated with aparticular gene sequence which has a high level of expression.Alternatively, promoters from mammalian expression products, such asactin, collagen, myosin, and the like, may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the gene sequences can bemodulated. Of interest are regulatory signals that aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or which are subject to chemical (such asmetabolite) regulation.

As discussed above, expression of the Δ4, 5 glycuronidase of theinvention in eukaryotic hosts is accomplished using eukaryoticregulatory regions. Such regions will, in general, include a promoterregion sufficient to direct the initiation of RNA synthesis. Preferredeukaryotic promoters include, for example, the promoter of the mousemetallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, Cell31:355-365 (1982)); the SV40 early promoter (Benoist et al., Nature(London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter(Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982);Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the Δ4, 5 glycuronidase of the inventiondoes not contain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading frame as the Δ4, 5 glycuronidase of the invention codingsequence) or a frame-shift mutation (if the AUG codon is not in the samereading frame as the Δ4, 5 glycuronidase of the invention codingsequence).

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay, for example, provide for prototrophy to an auxotrophic host or mayconfer biocide resistance to, e.g., antibiotics, heavy metals, or thelike. The selectable marker gene sequence can either be directly linkedto the DNA gene sequences to be expressed, or introduced into the samecell by co-transfection. Additional elements may also be needed foroptimal synthesis of the Δ4, 5 glycuronidase mRNA. These elements mayinclude splice signals, as well as transcription promoters, enhancers,and termination signals. cDNA expression vectors incorporating suchelements include those described by Okayama, Molec. Cell. Biol. 3:280(1983).

In a preferred embodiment, the introduced sequence will be incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, ColE1, pSC101,pACYC 184, and πVX). Such plasmids are, for example, disclosed bySambrook, et al. (Molecular Cloning: A Laboratory Manual, secondedition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring HarborLaboratory, 1989)). Bacillus plasmids include pC194, pC221, pT127, andthe like. Such plasmids are disclosed by Gryczan (In: The MolecularBiology of the Bacilli, Academic Press, NY (1982), pp. 307-329).Suitable Streptomyces plasmids include pIJ101 (Kendall et al., J.Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages suchas φC31 (Chater et al., In: Sixth International Symposium onActinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp.45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect.Dis. 8:693-704 (1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742(1978)).

Preferred eukaryotic plasmids include, for example, BPV, EBV, SV40,2-micron circle, and the like, or their derivatives. Such plasmids arewell known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274(1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces:Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold SpringHarbor, NY, p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon etal., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, In: CellBiology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression,Academic Press, NY, pp. 563-608 (1980)). Other preferred eukaryoticvectors are viral vectors. For example, and not by way of limitation,the pox virus, herpes virus, adenovirus and various retroviruses may beemployed. The viral vectors may include either DNA or RNA viruses tocause expression of the insert DNA or insert RNA.

Once the vector or DNA sequence containing the construct(s) has beenprepared for expression, the DNA construct(s) may be introduced into anappropriate host cell by any of a variety of suitable means, i.e.,transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,and the like. Additionally, DNA or RNA encoding the Δ4, 5 glycuronidaseof the invention may be directly injected into cells or may be impelledthrough cell membranes after being adhered to microparticles. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene sequence(s) results in the production ofthe Δ4, 5 glycuronidase of the invention. This can take place in thetransformed cells as such, or following the induction of these cells todifferentiate (for example, by administration of bromodeoxyuracil toneuroblastoma cells or the like).

The present invention also provides for the use of Δ4, 5 glycuronidaseas an enzymatic tool due to its substrate specificity and specificactivity. In a direct and more rigorous comparison between therecombinant and native enzymes, it was found that at least some of therecombinant enzyme)(Δ4,5^(Δ20) ) possessed at least about two-foldhigher and in some cases a roughly about three-fold higher specificactivity relative to the native Flavobacterial enzyme when measuredunder identical reaction conditions. Additionally, the activity of acloned enzyme is not compromised by its recombinant expression in E.coli.

The recombinant 44,5 glycuronidase exhibited a sharp ionic strengthdependence. These results are interesting given both the ionic characterof the disulfated heparin disaccharide used in the experiments describedbelow as well as the many ionic residues present within the enzyme thatmay function in substrate binding and/or catalysis; many of thesecharged residues are conserved in structurally and functionally relatedenzymes. From a substrate perspective, all of the unsaturateddisaccharides examined possess a negative charge (at pH 6.4) due to theC6 carboxylate of the uronic acid. It is possible that this acid acts asa critical structural determinant, especially given its proximity to theΔ4,5 bond. Charge neutralization of 6-O sulfate (e.g., in ΔUH_(NS,6S))could possibly be another contributing factor. From the enzymeperspective, the recombinant glycuronidase)(Δ4,5^(Δ20)) does possess 47basic residues (theoretical pI of 8.5), including R151 whose position isinvariantly conserved among the different glycuronidases examined. R151may possibly interact with the uronic acid carboxylate. At the sametime, Δ4,5 also possesses 44 acidic residues. At least ten of thesepositions are highly conserved. Charge masking of some of these ionicresidues (either acidic or basic) by increasing salt concentration mightinterfere with enzymatic activity. A similar observation of this ionicstrength dependency has been made for the heparinases [Ernst S, et alExpression in Escherichia coli, purification and characterization ofheparinase I from Flavobacterium heparinum. Biochem J. 1996 April 15;315 (pt 2): 589-97.]

A bell-shaped pH profile with a 6.4 optimum was also observed in thepresent invention. The 6.4 pH optimum generally agrees with resultsoriginally reported for the F. heparinum Δ4,5 as well as for more recentresults published for an unsaturated glucuronyl hydrolase purified fromBacillus sp. GL1 [Hashimoto, W., et al. (1999) Arch Biochem Biophys 368,367-74]. While there are 11 histidines present within the primarysequence, three histidines (H115, H201, and H218) appear to be highlyconserved. Interestingly, catalytically critical histidines also existin all three heparin lyases [Pojasek, K., et al. (2000) Biochemistry 39,4012-9] as well as chondroitin AC lyase [Huang, W., et al. (2001)Biochemistry 40, 2359-72] from Flavobacterium heparinum. While these twoclasses of enzymes cleave glycosaminoglycans by somewhat differentmechanisms (i.e., (β-elmination vs. hydrolysis), both would presumablyinvolve acid-base catalysis, viz the imidazole.

The question of substrate specificity has now been considered from threestructural perspectives: (1) the nature of the glycosidic linkage; (2)the relative sulfation pattern of the unsaturated disaccharide; and (3)the role of saccharide chain length (e.g., di- vs.

tetrasaccharide). Our results indicate that for the recombinant Δ4,5glycuronidase, there is an unambiguous preference for the 1→4 linkageover the 1→3 linkage making heparin rather than chondroitin/dermatanand/or hyaluronan the best substrate. It should be noted, however, thatwhile this linkage position is important, it is not absolute. Bothchondroitin and hyaluronan Δ4,5 disaccharides were hydrolyzed, albeit atmuch slower rates and using higher enzyme concentrations than wererequired to hydrolyze heparin disaccharides.

We also present a kinetic pattern of the Δ4,5 glycuronidase with regardto the specific sulfation within a heparin disaccharide. First andforemost, we find that unsaturated saccharides containing a 2-O-sulfateduronidate (ΔU_(2S)) at the non-reducing end are in general not cleavedby the Δ4,5 glycuronidase. Furthermore, the inability of a 2-O-sulfateddisaccharide to competitively inhibit the hydrolysis of non2-O-containing disaccharide substrates (such as ΔUH_(NAc)) furthersuggests that the presence of a 2-O sulfate precludes binding of thissaccharide to the enzyme.

In considering the effect of specific sulfate groups present on theglucosamine, the enzyme may be loosely summarized as having a gradedpreference for 6-O-sulfation but a clear selection against unsubstitutedor sulfated amines. This hierarchy is not an absolute distinction giventhe fact that all the non 2-O-containing heparin disaccharides examinedwere cleaved by the enzyme. Instead, it is based on relative kineticparameters. This apparent substrate discrimination at the N and 6positions of the glucosamine appears to be somewhat contextual,especially in the case of 6-O-sulfation. That is, while 6-0 sulfationmay bestow a favorable selectivity to a saccharide substrate, thispositive effect may be offset by the presence of a deacetylated amine(e.g., ΔUH_(NAc6S) vs. ΔUH_(NH26S) or ΔUH_(NS,6S)).

The structural preference the Δ4,5 demonstrates against 2-O-sulfateduronidates along with a so-called “N-position” discrimination for theglucosamine may be exploited for use of the glycuronidase as ananalytical tool for the compositional analyses of glycosaminoglycans. Wewere able to predict the extent and relative rates by which specificdisaccharide “peaks” would disappear (i.e., due to theglycuronidase-dependent loss of absorbance at 232 nm.), based entirelyon our kinetically defined substrate specificity determinationsdescribed in the Examples below. All 2-O-sulfate containingdisaccharides tested were refractory to hydrolysis by the Δ4,5glycuronidase. On the other hand, the remaining disaccharides werehydrolyzed in a time-dependent fashion that corresponded to theirrelative substrate specificities (i.e.,ΔUH_(NAc6S)>ΔUH_(NS,6S)>ΔUH_(NS)).

From this experiment, another important and surprising observation wasmade, namely that the Δ4,5 glycuronidase also hydrolyzes Δ4,5unsaturated tetrasaccharides. It is also very interesting to note thatthis particular tetrasaccharide is as good of a substrate as thedisaccharide ΔUH_(NS). This observation may argue against a substratediscrimination used by the enzyme that is negatively based on increasingmolecular weight as was first reported [Hovingh, P. and Linker, A.(1977) Biochem J 165, 287-93].

Therefore, the invention also provides for the cleavage ofglycosaminoglycans using the substantially pure Δ4,5 glycuronidasedescribed herein. The Δ4,5 glycuronidase of the invention may be used tospecifically cleave an HSGAG by contacting the HSGAG substrate with theΔ4,5 glycuronidase of the invention. The invention is useful in avariety of in vitro, in vivo and ex vivo methods in which it is usefulto cleave HSGAGs.

As used herein the terms “HSGAG”, “GAG”, and “glycosaminoglycans” areused interchangeably to refer to a family of molecules havingheparin-like/heparan sulfate-like structures and properties. Thesemolecules include but are not limited to low molecular weight heparin(LMWH), heparin, biotechnologically prepared heparin, chemicallymodified heparin, synthetic heparin, and heparan sulfate. The term“biotechnological heparin” encompasses heparin that is prepared fromnatural sources of polysaccharides which have been chemically modifiedand is described for example in Razi et al., Bioche. J. 1995 Jul 15;309(Pt 2): 465-72. Chemically modified heparin is described in Yates etal., Carbohydrate Res (1996) Nov 20;294:15-27, and is known to those ofskill in the art. Synthetic heparin is well known to those of skill inthe art and is described in Petitou, M. et al., Bioorg Med Chem Lett.(1999) Apr 19;9(8):1161-6.

Analysis of a sample of glycosaminoglycans is also possible with Δ4,5glycuronidase alone or in conjunction with other enzymes. Other HSGAGdegrading enzymes include but are not limited to heparinase-I,heparinase- II , heparinase-III, heparinase-IV, D-glucuronidase andL-iduronidase, modified versions of heparinases, variants andfunctionally active fragments thereof.

The methods that may be used to test the specific activity of Δ4,5glycuronidase of the present invention are known in the art, e.g., thosedescribed in the Examples. These methods may also be used to assess thefunction of variants and functionally active fragments of Δ4,5glycuronidase. The k_(cat) value may be determined using any enzymaticactivity assay to assess the activity of a Δ4,5 glycuronidase enzyme.Several such assays are well-known in the art. For instance, an assayfor measuring k_(cat) is described in (Ernst, S. E., Venkataraman, G.,Winkler, S., Godavarti, R., Langer, R., Cooney, C. and Sasisekharan. R.(1996) Biochem. J. 315, 589-597. The “native Δ4,5 glycuronidase katvalue” is the measure of enzymatic activity of the native Δ4,5glycuronidase obtained from cell lysates of F. heparinum also describedin the Examples below. Therefore, based on the disclosure providedherein, those of ordinary skill in the art will be able to identifyother Δ4,5 glycuronidase molecules having altered enzymatic activitywith respect to the naturally occurring Δ4,5 glycuronidase molecule suchas functional variants. The term “specific activity” as used hereinrefers to the enzymatic activity of a preparation of Δ4,5 glycuronidase.In general, it is preferred that the substantially pure and/or isolatedΔ4,5 glycuronidase preparations of the invention have a specificactivity of at least about 60 picomoles of substrate hydrolized perminute per picomole of enzyme. This generally corresponds to a k_(cat)of at least about 10 per second for the enzyme using a substrate such asheparin disaccharide ΔUH_(NAc).

Due to the activity of Δ4,5 glycuronidase on glycosaminoglycans, theproduct profile produced by a Δ4,5 glycuronidase may be determined byany method known in the art for examining the type or quantity ofdegradation product produced by Δ4,5 glycuronidase alone or incombination with other enzymes. One of skill in the art will alsorecognize that the Δ4,5 glycuronidase may also be used to assess thepurity of glycosaminoglycans in a sample. One preferred method fordetermining the type and quantity of product is described in Rhomberg,A. J. et al., PNAS, v. 95, p. 4176-4181, (April 1998), which is herebyincorporated in its entirety by reference. The method disclosed in theRhomberg reference utilizes a combination of mass spectrometry andcapillary electrophoretic techniques to identify the enzymatic productsproduced by heparinase. The Rhomberg study utilizes heparinase todegrade HSGAGs to produce HSGAG oligosaccharides. MALDI (Matrix-AssistedLaser Desorption Ionization) mass spectrometry can be used for theidentification and semiquantitative measurement of substrates, enzymes,and end products in the enzymatic reaction. The capillaryelectrophoresis technique separates the products to resolve even smalldifferences amongst the products and is applied in combination with massspectrometry to quantitate the products produced. Capillaryelectrophoresis may even resolve the difference between a disaccharideand its semicarbazone derivative. Detailed methods for sequencingpolysaccharides and other polymers are disclosed in co-pending U.S.Patent Applications Serial Nos. 09/557,997 and 09/558,137, both filed onApril 24, 2000 and having common inventorship. The entire contents ofboth applications are hereby incorporated by reference. For example, themethod is performed by enzymatic digestion, followed by massspectrometry and capillary electrophoresis. The enzymatic assays can beperformed in a variety of manners, as long as the assays are performedidentically on the Δ4,5 glycuronidase, so that the results may becompared. In the example described in the Rhomberg reference, enzymaticreactions are performed by adding 1 mL of enzyme solution to 5 mL ofsubstrate solution. The digestion is then carried out at roomtemperature (22° C.), and the reaction is stopped at various time pointsby removing 0.5 mL of the reaction mixture and adding it to 4.5 mL of aMALDI matrix solution, such as caffeic acid (approximately 12 mg/mL) and70% acetonitrile/water. The reaction mixture is then subjected to MALDImass spectrometry. The MALDI surface is prepared by the method of Xiangand Beavis (Xiang and Beavis (1994) Rapid. Commun. Mass. Spectrom. 8,199-204). A two-fold lower access of basic peptide (Arg/Gly)l₅ ispremixed with matrix before being added to the oligosaccharide solution.A 1 mL aliquot of sample/matrix mixture containing 1-3 picomoles ofoligosaccharide is deposited on the surface. After crystallizationoccurs (typically within 60 seconds), excess liquid is rinsed off withwater. MALDI mass spectrometry spectra is then acquired in the linearmode by using a PerSeptive Biosystems (Framingham, MA) Voyager Elitereflectron time-of-flight instrument fitted with a 337 nanometernitrogen laser. Delayed extraction is used to increase resolution (22kV, grid at 93%, guidewire at 0.15%, pulse delay 150 ns, low mass gateat 1,000, 128 shots averaged). Mass spectra are calibrated externally byusing the signals for proteinated (Arg/Gly)l₅ and its complex with theoligosaccharide.

Capillary electrophoresis may then be performed on aHewlett-Packard^(3D) CE unit by using uncoated fused silica capillaries(internal diameter 75 micrometers, outer diameter 363 micrometers,1_(det) 72.1 cm, and l_(tot) 85 cm). Analytes are monitored by using UVdetection at 230 nm and an extended light path cell (Hewlett-Packard).The electrolyte is a solution of 10 mL dextran sulfate and 50 millimolarTris/phosphoric acid (pH2.5). Dextran sulfate is used to suppressnonspecific interactions of the heparin oligosaccharides with a silicawall. Separations are carried out at 30 kV with the anode at thedetector side (reversed polarity). A mixture of a1/5-naphtalenedisulfonic acid and 2-naphtalenesulfonic acid (10micromolar each) is used as an internal standard.

Other methods for assessing the product profile may also be utilized.For instance, other methods include methods which rely on parameterssuch as viscosity (Jandik, K. A., Gu, K. and Linhardt, R. J., (1994),Glycobiology, 4:284-296) or total UV absorbance (Ernst,

S. et al., (1996), Biochem. J., 315:589-597) or mass spectrometry orcapillary electrophoresis alone.

The Δ4,5 glycuronidase molecules of the invention are also useful astools for sequencing HSGAGs. Detailed methods for sequencingpolysaccharides and other polymers are disclosed in co-pending U.S.Patent Applications Serial Nos. 09/557,997 and 09/558,137, both filed onApril 24, 2000 and having common inventorship. These methods utilizetools such as heparinases in the sequencing process. The Δ4,5glycuronidase of the invention is useful as such a tool.

One of ordinary skill in the art, in light of the present disclosure, isenabled to produce substantially pure preparations of HSGAG and/or GAGfragment compositions utilizing the Δ4, 5 glycuronidase molecules aloneor in conjunction with other enzymes.

These GAG fragments have many therapeutic utilities. The glycuronidasemolecules and/or GAG fragments can be used for the treatment of any typeof condition in which GAG fragment therapy has been identified as auseful therapy, e.g., preventing coagulation, inhibiting angiogenesis,inhibiting proliferation. The GAG fragment preparations are preparedfrom HSGAG sources. A “HSGAG source” as used herein refers toheparin-like/heparan sulfate-like glycosaminoglycan composition whichcan be manipulated to produce GAG fragments using standard technology,including enzymatic degradation etc. As described above, HSGAGs includebut are not limited to isolated heparin, chemically modified heparin,biotechnology prepared heparin, synthetic heparin, heparan sulfate, andLMWH. Thus HSGAGs can be isolated from natural sources, prepared bydirect synthesis, mutagenesis, etc.

Thus, the methods of the invention enable one of skill in the art toprepare or identify an appropriate composition of GAG fragments,depending on the subject and the disorder being treated. Thesecompositions of GAG fragments may be used alone or in combination withthe Δ4,5 glycuronidase and/or other enzymes. Likewise Δ4, 5glycuronidase and/or other enzymes may also be used to produce GAGfragments in vivo.

The compositions of the invention can be used for the treatment of anytype of condition in which GAG fragment therapy has been identified as auseful therapy. Thus, the invention is useful in a variety of in vitro,in vivo and ex vivo methods in which therapies are useful. For instance,it is known that GAG fragments are useful for preventing coagulation,inhibiting cancer cell growth and metastasis, preventing angiogenesis,preventing neovascularization, preventing psoriasis. The GAG fragmentcompositions may also be used in in vitro assays, such as a qualitycontrol sample.

Each of these disorders is well-known in the art and is described, forinstance, in Harrison's Principles of Internal Medicine (McGraw Hill,Inc., New York), which is incorporated by reference.

In one embodiment the preparations of the invention are used forinhibiting angiogenesis. An effective amount for inhibiting angiogenesisof the GAG fragment preparation is administered to a subject in need oftreatment thereof. Angiogenesis as used herein is the inappropriateformation of new blood vessels. “Angiogenesis” often occurs in tumorswhen endothelial cells secrete a group of growth factors that aremitogenic for endothelium causing the elongation and proliferation ofendothelial cells which results in a generation of new blood vessels.Several of the angiogenic mitogens are heparin binding peptides whichare related to endothelial cell growth factors. The inhibition ofangiogenesis can cause tumor regression in animal models, suggesting ause as a therapeutic anticancer agent. An effective amount forinhibiting angiogenesis is an amount of GAG fragment preparation whichis sufficient to diminish the number of blood vessels growing into atumor. This amount can be assessed in an animal model of tumors andangiogenesis, many of which are known in the art.

The Δ4, 5 glycuronidase is, in some embodiments, immobilized on asupport. The glycuronidase may be immobilized to any type of support butif the support is to be used in vivo or ex vivo it is desired that thesupport is sterile and biocompatible. A biocompatible support is onewhich would not cause an immune or other type of damaging reaction whenused in a subject. The Δ4, 5 glycuronidase may be immobilized by anymethod known in the art. Many methods are known for immobilizingproteins to supports. A “solid support” as used herein refers to anysolid material to which a polypeptide can be immobilized.

Solid supports, for example, include but are not limited to membranes,e.g., natural and modified celluloses such as nitrocellulose or nylon,Sepharose, Agarose, glass, polystyrene, polypropylene, polyethylene,dextran, amylases, polyacrylamides, polyvinylidene difluoride, otheragaroses, and magnetite, including magnetic beads. The carrier can betotally insoluble or partially soluble and may have any possiblestructural configuration. Thus, the support may be spherical, as in abead, or cylindrical, as in the inside surface of a test tube ormicroplate well, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, bottom surface of amicroplate well, etc. The Δ4, 5 glycuronidase of the invention may alsobe used to remove active GAGs from a GAG containing fluid. A GAGcontaining fluid is contacted with the Δ4, 5 glycuronidase of theinvention to degrade the GAG. The method is particularly useful for theex vivo removal of GAGs from blood. In one embodiment of the inventionthe Δ4, 5 glycuronidase is immobilized on a solid support as isconventional in the art. The solid support containing the immobilizedΔ4, 5 glycuronidase may be used in extracorporeal medical devices (e.g.hemodialyzer, pump-oxygenator) for systemic heparinization to preventthe blood in the device from clotting. The support membrane containingimmobilized Δ4, 5 glycuronidase is positioned at the end of the deviceto neutralize the GAG before the blood is returned to the body.

Thus, the Δ4, 5 glycuronidase molecules are useful for treating orpreventing disorders associated with coagulation. A “disease associatedwith coagulation” as used herein refers to a condition characterized byan interruption in the blood supply to a tissue due to a blockage of theblood vessel responsible for supplying blood to the tissue such as isseen for myocardial or cerebral infarction. A cerebral ischemic attackor cerebral ischemia is a form of ischemic condition in which the bloodsupply to the brain is blocked. This interruption in the blood supply tothe brain may result from a variety of causes, including an intrinsicblockage or occlusion of the blood vessel itself, a remotely originatedsource of occlusion, decreased perfusion pressure or increased bloodviscosity resulting in inadequate cerebral blood flow, or a rupturedblood vessel in the subarachnoid space or intracerebral tissue.

The Δ4, 5 glycuronidase or the GAG fragments generated therewith may beused alone or in combination with a therapeutic agent for treating adisease associated with coagulation. Examples of therapeutics useful inthe treatment of diseases associated with coagulation includeanticoagulation agents, antiplatelet agents, and thrombolytic agents.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation. Anticoagulants include, but are not limitedto, heparin, warfarin, coumadin, dicumarol, phenprocoumon,acenocoumarol, ethyl biscoumacetate, and indandione derivatives.

Antiplatelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack or stroke. Antiplatelet agents include, butare not limited to, aspirin, thienopyridine derivatives such asticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well asRGD mimetic s and also antithrombin agents such as, but not limited to,hirudin. Thrombolytic agents lyse clots which cause the thromboembolicstroke. Thrombolytic agents have been used in the treatment of acutevenous thromboembolism and pulmonary emboli and are well known in theart (e.g. see Hennekens et al, J Am Coll Cardiol; v. 25 (7 supp), p.18S-22S (1995); Holmes, et al, J Am Coll Cardiol; v.25 (7 supply, p.10S-17S(1995)). Thrombolytic agents include, but are not limited to,plasminogen, a₂-antiplasmin, streptokinase, antistreplase, tissueplasminogen activator (tPA), and urokinase. “tPA” as used hereinincludes native tPA and recombinant tPA, as well as modified forms oftPA that retain the enzymatic or fibrinolytic activities of native tPA.The enzymatic activity of tPA can be measured by assessing the abilityof the molecule to convert plasminogen to plasmin. The fibrinolyticactivity of tPA may be determined by any in vitro clot lysis activityknown in the art, such as the purified clot lysis assay described byCarlson, et. al., Anal. Biochem. 168, 428-435 (1988) and its modifiedform described by Bennett, W. F. et al., 1991, J. Biol. Chem.266(8):5191-5201, the entire contents of which are hereby incorporatedby reference. The invention compositions of the invention are useful forthe same purposes as heparinases and the degradation products ofheparinases (HSGAG fragments). Thus, for instance, the compositions ofthe invention are useful for treating and preventing cancer cellproliferation and metastasis. Thus, according to another aspect of theinvention, there is provided methods for treating subjects having or atrisk of having cancer. Critically, HSGAGs (along with collagen) are keycomponents of the cell surface-extracellular matrix (ECM) interface.While collagen-like proteins provide the necessary extracellularscaffold for cells to attach and form tissues, the complexpolysaccharides fill the space created by the scaffold and act as amolecular sponge by specifically binding and regulating the biologicalactivities of numerous signaling molecules like growth factors,cytokines etc. It has recently been recognized that cells synthesizedistinct HSGAG sequences and decorate themselves with these sequences,using the extraordinary information content present in the sequences tobind specifically to many signaling molecules and thereby regulatevarious biological processes.

The invention also contemplates the use of therapeutic GAG fragments forthe treatment and prevention of tumor cell proliferation and metastasis.A “therapeutic GAG fragment” as used herein refers to a molecule ormolecules which are pieces or fragments of a GAG that have beenidentified or generated through the use of the Δ4, 5 glycuronidasepossibly along with other naturally occurring and/or modifiedheparinases. In some aspects the therapeutic GAG fragments have the samestructure as commercially available LMWH, but are generated using theΔ4, 5 glycuronidase.

The invention also encompasses screening assays for identifyingtherapeutic GAG fragments for the treatment of a tumor and forpreventing metastasis. The assays are accomplished by treating a tumoror isolated tumor cells with 04, 5 glycuronidase and/or other naturallyoccurring or modified heparinases and isolating the resultant GAGfragments. Surprisingly, these GAG fragments have therapeutic activityin the prevention of tumor cell proliferation and metastasis. Thus theinvention encompasses individualized therapies, in which a tumor orportion of a tumor is isolated from a subject and used to prepare thetherapeutic GAG fragments. These therapeutic fragments can bere-administered to the subject to protect the subject from further tumorcell proliferation or metastasis or from the initiation of metastasis ifthe tumor is not yet metastatic. Alternatively the fragments can be usedin a different subject having the same type or tumor or a different typeof tumor.

Therapeutic GAG fragments include GAG fragments which have therapeuticactivity in that they prevent the proliferation and/or metastasis of atumor cell. Such compounds may be generated using 04, 5 glycuronidase toproduce therapeutic fragments or they may be synthesized de novo.Putative GAG fragments can be tested for therapeutic activity using anyof the assays described herein or known in the art. Thus the therapeutic

GAG fragment may be a synthetic GAG fragment generated based on thesequence of the GAG fragment identified when the tumor is contacted withΔ4, 5 glycuronidase, or having minor variations which do not interferewith the activity of the compound. Alternatively the therapeutic GAGfragment may be an isolated GAG fragment produced when the tumor iscontacted with Δ4, 5 glycuronidase.

The invention is useful for treating and/or preventing tumor cellproliferation or metastasis in a subject. The terms “treat” and“treating” tumor cell proliferation as used herein refer to inhibitingcompletely or partially the proliferation or metastasis of a cancer ortumor cell, as well as inhibiting any increase in the proliferation ormetastasis of a cancer or tumor cell.

A “subject having a cancer” is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas.

A “subject at risk of having a cancer” as used herein is a subject whohas a high probability of developing cancer. These subjects include, forinstance, subjects having a genetic abnormality, the presence of whichhas been demonstrated to have a correlative relation to a higherlikelihood of developing a cancer and subjects exposed to cancer causingagents such as tobacco, asbestos, or other chemical toxins, or a subjectwho has previously been treated for cancer and is in apparent remission.When a subject at risk of developing a cancer is treated with a Δ4, 5glycuronidase or degradation product thereof the subject may be able tokill the cancer cells as they develop. Effective amounts of the Δ4, 5glycuronidase, variant 44, 5 glycuronidase or therapeutic GAGs of theinvention are administered to subjects in need of such treatment.Effective amounts are those amounts which will result in a desiredimprovement in the condition or symptoms of the condition, e.g., forcancer this is a reduction in cellular proliferation or metastasis,without causing other medically unacceptable side effects. Such amountscan be determined with no more than routine experimentation. It isbelieved that doses ranging from 1 nanogram/kilogram to 100milligrams/kilogram, depending upon the mode of administration, will beeffective. The absolute amount will depend upon a variety of factors(including whether the administration is in conjunction with othermethods of treatment, the number of doses and individual patientparameters including age, physical condition, size and weight) and canbe determined with routine experimentation. It is preferred generallythat a maximum dose be used, that is, the highest safe dose according tosound medical judgment. The mode of administration may be any medicallyacceptable mode including oral, subcutaneous, intravenous, etc.

In some aspects of the invention the effective amount of Δ4, 5glycuronidase or therapeutic GAG is that amount effective to preventinvasion of a tumor cell across a barrier. The invasion and metastasisof cancer is a complex process which involves changes in cell adhesionproperties which allow a transformed cell to invade and migrate throughthe extracellular matrix (ECM) and acquire anchorage-independent growthproperties Liotta, L. A., et al., Cell 64:327-336, 1991. Some of thesechanges occur at focal adhesions, which are cell/ECM contact pointscontaining membrane-associated, cytoskeletal, and intracellularsignaling molecules. Metastatic disease occurs when the disseminatedfoci of tumor cells seed a tissue which supports their growth andpropagation, and this secondary spread of tumor cells is responsible forthe morbidity and mortality associated with the majority of cancers.Thus the term “metastasis” as used herein refers to the invasion andmigration of tumor cells away from the primary tumor site.

The barrier for the tumor cells may be an artificial barrier in vitro ora natural barrier in vivo. In vitro barriers include but are not limitedto extracellular matrix coated membranes, such as Matrigel. Thus the Δ4,5 glycuronidase compositions or degradation products thereof can betested for their ability to inhibit tumor cell invasion in a Matrigelinvasion assay system as described in detail by Parish, C.R., et al., “ABasement-Membrane Permeability Assay which Correlates with theMetastatic Potential of Tumour Cells,” Int. J. Cancer, 1992, 52:378-383.Matrigel is a reconstituted basement membrane containing type

IV collagen, laminin, heparan sulfate proteoglycans such as perlecan,which bind to and localize bFGF, vitronectin as well as transforminggrowth factor-β (TGF-β), urokinase-type plasminogen activator (uPA),tissue plasminogen activator (tPA), and the serpin known as plasminogenactivator inhibitor type 1 (PAI-1). Other in vitro and in vivo assaysfor metastasis have been described in the prior art, see, e.g., U.S.Pat. No. 5,935,850, issued on Aug. 10, 1999, which is incorporated byreference. An in vivo barrier refers to a cellular barrier present inthe body of a subject.

In general, when administered for therapeutic purposes, the formulationsof the invention are applied in pharmaceutically acceptable solutions.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, adjuvants, and optionally other therapeutic ingredients.

The compositions of the invention may be administered per se (neat) orin the form of a pharmaceutically acceptable salt. When used in medicinethe salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V);and phosphoric acid and a salt (0.8-2% W/V). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9%W/V); parabens (0.01-0.25% WN) and thimerosal (0.004-0.02% W/V).

The present invention provides pharmaceutical compositions, for medicaluse, which comprise Δ4, 5 glycuronidase, variant Δ4, 5 glycuronidase ofthe invention, or therapeutic GAG fragments together with one or morepharmaceutically acceptable carriers and optionally other therapeuticingredients. The term “pharmaceutically-acceptable carrier” as usedherein, and described more fully below, means one or more compatiblesolid or liquid filler, dilutants or encapsulating substances which aresuitable for administration to a human or other animal. In the presentinvention, the term “carrier” denotes an organic or inorganicingredient, natural or synthetic, with which the active ingredient iscombined to facilitate the application. The components of thepharmaceutical compositions also are capable of being commingled withthe Δ4, 5 glycuronidase of the present invention or other compositions,and with each other, in a manner such that there is no interaction whichwould substantially impair the desired pharmaceutical efficiency.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular active agentselected, the particular condition being treated and the dosage requiredfor therapeutic efficacy. The methods of this invention, generallyspeaking, may be practiced using any mode of administration that ismedically acceptable, meaning any mode that produces effective levels ofthe drug without causing clinically unacceptable adverse effects. Apreferred mode of administration is a parenteral route. The term“parenteral” includes subcutaneous injections, intravenous,intramuscular, intraperitoneal, intra sternal injection or infusiontechniques. Other modes of administration include oral, mucosal, rectal,vaginal, sublingual, intranasal, intratracheal, inhalation, ocular,transdermal, etc.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active Δ4, 5 glycuronidaseinto association with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the polymer into association with a liquid carrier,a finely divided solid carrier, or both, and then, if necessary, shapingthe product. The polymer may be stored lyophilized.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the heparinases of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polylactic andpolyglycolic acid, polyanhydrides and polycaprolactone; nonpolymersystems that are lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono-, di andtriglycerides; hydrogel release systems; silastic systems; peptide basedsystems; wax coatings, compressed tablets using conventional binders andexcipients, partially fused implants and the like. Specific examplesinclude, but are not limited to: (a) erosional systems in which thepolysaccharide is contained in a form within a matrix, found in U.S.Pat. Nos. 4,452,775 (Kent); 4,667,014 (Nestor et al.); and 4,748,034 and5,239,660 (Leonard) and (b) diffusional systems in which an activecomponent permeates at a controlled rate through a polymer, found inU.S. Pat. Nos. 3,832,253 (Higuchi et al.) and 3,854,480 (Zaffaroni). Inaddition, a pump-based hardware delivery system can be used, some ofwhich are adapted for implantation.

A subject is any human or non-human vertebrate, e.g., dog, cat, horse,cow, pig.

When administered to a patient undergoing cancer treatment, the Δ4, 5glycuronidase or therapeutic GAG compounds may be administered incocktails containing other anti-cancer agents. The compounds may also beadministered in cocktails containing agents that treat the side-effectsof radiation therapy, such as anti-emetics, radiation protectants, etc.

Anti-cancer drugs that can be co-administered with the compounds of theinvention include, but are not limited to Acivicin; Aclarubicin;Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;

Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine;

Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin;Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin;Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate;Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine

Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine;Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone;Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea;Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a;Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; InterferonBeta-I a; Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride;Lanreotide Acetate; Letrozole; Leuprolide Acetate; LiarozoleHydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride;Masoprocol; Maytansine; Mechlorethamine Hydrochloride; MegestrolAcetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine;Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide;Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper;Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole;Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan;Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium;Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin;Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol;Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin;Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan Sodium;Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone;Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin;Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; TrestoloneAcetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate;Triptorelin; Tubulozole Hydrochloride; Uracil

Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate;Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate;Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate;Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;Zinostatin; Zorubicin Hydrochloride.

The Δ4, 5 glycuronidase or therapeutic GAG compounds may also be linkedto a targeting molecule. A targeting molecule is any molecule orcompound which is specific for a particular cell or tissue and which canbe used to direct the Δ4, 5 glycuronidase or therapeutic GAG to the cellor tissue. Preferably the targeting molecule is a molecule whichspecifically interacts with a cancer cell or a tumor. For instance, thetargeting molecule may be a protein or other type of molecule thatrecognizes and specifically interacts with a tumor antigen.

Tumor-antigens include Melan-A/M-ART-1, Dipeptidyl peptidase IV (DPPIV),adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectalassociated antigen (CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA)and its immunogenic epitopes CAP-1 and CAP-2, etv6, am11, ProstateSpecific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, andPSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-Δ4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100^(Pmel117), PRAME, NY-ESO-1, brain glycogen phosphorylase, SSX-1,SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27, adenomatouspolyposis coli protein (APC), fodrin, P1A, Connexin 37, Ig-idiotype,p15, gp75, GM2 and GD2 gangliosides, viral products such as humanpapilloma virus proteins, Smad family of tumor antigens, lmp-1,EBV-encoded nuclear antigen (EBNA)-1, and c-erbB-2.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Materials and Methods

Chemicals and reagents. Unless otherwise stated, biochemicals werepurchased from Sigma Aldrich Chemical (St. Louis, Mo.). Disaccharideswere purchased from Calbiochem (San Diego, Calif.). Reagents for λZAP IIgenomic library construction and screening were obtained from Stratagene(La Jolla, Calif.). Restriction endonucleases were purchased from NewEngland Biolabs (Beverly, Ma.). DNA oligonucleotide primers weremanufactured by Invitrogen/Life Technologies (Carlsbad, Calif.).Additional molecular cloning reagents were obtained from themanufacturers listed.

Bacterial strains and growth conditions. F. heparinum (Pedobacterheparinus) was obtained as a lyophilized stock from American TypeCulture Collection (ATCC, Manassas, Va.), stock no. 13125. Rehydratedcultures were grown aerobically at 30° C. with moderate shaking to anoptical density (A₆₀₀) between 1.5 and 2 in defined media containing 6.4mM NaH₂PO₄, 7.6 mM Na₂HPO₄, 12 mM KH₂PO₄, 14.3 mM K₂HPO₄, 1.7 mM NaCl,and 1.9 mM NH₄Cl, pH 6.9 and supplemented with 0.1 mM trace metalsCaCl₂, FeSO₄, CuSO₄, NaMoO₄, CoCl₂, and MnSO₄ (added from a 100× stockin 10 mM H₂SO₄), 0.8% glucose, 0.05% methionine, 0.05% histidine, 2 mMMgSO₄, and 0.1% heparin all added under sterile conditions. E. colistrains included TOP10 (Invitrogen) or DH5α for PCR cloning andsubcloning and BL21(DE3) (Novagen, Madison Wis.) for recombinant proteinexpression. Bacteriophage host strains XL1-Blue MRF' and SOLR wereobtained from Stratagene.

Purification of glycuronidase peptides and protein sequencing. The 4, 5glycuronidase was purified from 10 liter fermentation cultures using amethod such as those described in McLean, M. W., Bruce, J. S., Long, W.F., and Williamson, F. B., 1984, Eur J Biochem 145, 607-15.

Molecular cloning of the Δ4, 5 glycuronidase gene from F. heparinumgenomic DNA.

Flavobacterial genomic DNA was isolated from 10 mL of Flavobacterialculture using the QIAGEN DNeasy DNA purification kit according to theManufacturer's instructions for gram-negative bacteria usingapproximately 2×10⁹ cells per column. Following purification, genomicDNA was ethanol precipitated and resuspended in TE, pH 7.5 at 0.5 mg/mL.The quality of genomic DNA was confirmed spectrophotometrically at260/280 nm, by electrophoresis on a 0.5% agarose gel and by PCR usingFlavobacterial specific primers.

The following degenerate primers were synthesized from peptidescorresponding initially to peaks 19 and 24 (Example 1): 5′GARACNCAYCARGGNYTNACNAAYGAR 3′ (SEQ ID NO. 5) (peak 19 forward), 5′YTCRTTNGTNARNCCYTGRTGNGTYTC 3′ (SEQ ID NO. 6) (peak 19 reverse); 5′AAYTAYGCNGAYTAYTAYTAY 3′ (SEQ ID NO. 7) (peak 24 forward); 5′RTARTARTARTCNGCRTARTT 3′ (SEQ ID NO. 8) (peak 24 reverse). All fourprimers were screened in a PCR assay using all possible pairings(forward and reverse). The PCR reaction conditions included 200 ng ofgenomic DNA, 200 picomoles for each forward and reverse primer, 200 μMdNTPs, 1 unit of Vent DNA Polymerase (New England Biolabs) in a 100_(j—)d reaction volume. 35 cycles were completed using a 52° C.annealing temperature and 1.5 minute extensions at 72° C. The 450 byproduct amplified using primers 19 forward and 24 reverse was gelpurified and subject to direct

DNA sequencing which confirmed the inclusion of translated sequencecorresponding to peptide peaks 19 and 24 in addition to peak 12. Thesame DNA was also ³²P radiolabeled by random priming using 200 μCiα³²P[dCTP] at 6000 Ci/mmole (NEN, Boston, Ma.), 50-100 ng of DNA and thePrime-it II random priming kit (Stratagene) (probel). Unincorporated ³²PdNTPs were removed by gel filtration using G-50 Quick-spin columns(Roche Biochemicals, N.J.). Labeling reactions typically yieldedapproximately 50 ng of radiolabeled DNA with specific activitiesexceeding 10⁹ cpm/μg.

DNA hybridization probe 2 was initially created by PCR as describedabove except using degenerate primer 26 5′ CARACNTAYACNCCNGGNATGAAY 3′(SEQ ID NO. 9) (peak 26 forward) and 20 picomoles of reverse,non-degenerate primer 54 (5′ TTCATGGTCGTAACCGCATG 3′) (SEQ ID NO. 10);the latter oligonucleotide corresponds to Δ4, 5 DNA sequence 3′ of peak8. Direct sequencing of this PCR fragment confirmed the presence of peak26 and peak 13 peptides. DNA probe 3 used in DNA southern hybridizations(below) was PCR amplified from genomic DNA using primer 68 (5′TATACACCAGGCATGAACCC 3′) (SEQ ID NO. 11) and 74 (5′CCCAGTATAAATACTCCAGGT 3′) (SEQ ID NO. 12).

Plaque hybridization screening of F. heparinum genomic library. A λ ZAPII genomic library (Stratagene) was constructed as described[Sasisekharan, R., Bulmer, M., Moremen, K. W., Cooney, C. L., andLanger, R. (1993) Proc Natl Acad Sci USA 90, 3660-4]. The amplifiedlibrary (1×10¹⁰ pfu/mL) was plated out at approximately 1×10⁶ pfu(50,000 pfu/plate) on 100×150 mm LB plates. Plaque lifts on to nylonmembranes (Nytran Supercharge, Schleicher and Schuell) and subsequenthybridization screenings were completed using standard methods andsolutions [Current Protocols in Molecular Biology,1987 , John Wiley andSons, New York]. DNA was crosslinked to each filter by UV-irradiation(Stratagene Stratalinker) for 30 seconds at 1200 joules/cm³.Hybridizations were carried at 42° C. using 10⁷-10⁸ cpm of radiolabeledprobe (at approximately 0.25 ng/mL). Low stringency washes were carriedout at room temperature in 2× SSC, 0.1% SDS; high stringency washescarried out at 58-60° C. in 0.2× SSC and 0.1% SDS. Hybridized plaqueswere visualized by phosphor imaging (Molecular Dynamics) and/or ³²Pautoradiography. Tertiary screens of positive clones were completed andthe recombinant phage was excised as a double-stranded phagemid(pBluescript) using the ExAssist interference-resistant helper phage andSOLR strain according to the manufacturer's protocol (Stratagene).Recombinants were characterized by DNA sequencing using both T7 and T3primers.

Creation of a flavobacterium Bgl II-EcoR1 subgenomic library forisolation of the Δ4, 5 5′ terminus. 1 μg of genomic DNA was cut with 20units of Eco R1, Bgl II, and Hind III individually or as double digests.Restriction products were resolved by gel electrophoresis on 1 agarosegels run in 1× TAE buffer. Southern DNA hybridizations were completedaccording to standard protocols [Current Protocols in Molecular Biology,1987, John Wiley and Sons, New York] using ³²P radiolabeled probe 3.Based on this Southern analysis, 5 μg of Flavobacterial genomic DNA wasdigested with Bgl-II-Eco R1 and the DNA resolved on a preparative 1%agarose gel run under identical conditions as those described for theanalytical gel. DNA ranging from approximately 1-2 kb was gel purifiedand ligated into pLITMUS as a Bgl II-EcoR1 cassette. Positive cloneswere identified by PCR colony screening using primers 68 and 74 andconfirmed by DNA sequencing.

PCR cloning of Δ4, 5 gene and recombinant expression in E. coli. Thefull-length glycuronidase gene was directly PCR amplified from genomicDNA using forward primer 85 5′ TGTTCTAGACATATGAAATCACTACTCAGTGC (SEQ IDNO. 13) 3′ and reverse primer 86 5′ GTCTCGAGGATCCTTAAGACTGATTAATTGTT 3′(SEQ ID NO. 14) (with Nde 1 and Xho1 restriction sites denoted in bold),200 ng genomic DNA, and Vent DNA Polymerase for 35 cycles. dA overhangswere generated in a final 10 minute extension at 72° C. using AmpliTaqDNA polymerase (Applied Biosystems). PCR products were gel purified,ligated into the TOPO/TA PCR cloning vector (Invitrogen), andtransformed into One-shot TOP10 chemically competent cells. Positiveclones were identified by blue/white colony selection and confirmed byPCR colony screening. The 1.2 kb Δ4, 5 gene was subcloned intoexpression plasmid pET28a (Novagen) as an Nde 1-Xho 1 cassette. Finalexpression clones were confirmed by plasmid DNA sequencing.

For the expression of Δ4, 5 glycuronidase beginning with M21)(Δ4,5²⁰ ⁾,the forward primer 95 5′ TGT TCT AGA CAT ATG ACA GTT ACG AAA GGC AA 3′(SEQ ID NO. 15) (also containing an Nde 1 restriction site near its 5′terminus) was used in place of primer 85 (above). 50 ng of the originalexpression plasmid pET28a/Δ4, 5 was used as the DNA template in PCRreactions involving a total of 20 cycles. Otherwise, cloning was asdescribed for the full-length gene. Both pET28a/Δ4, 5 and pET28aΔ4,5²⁰plasmids were transformed into BL21 (DE3) for expression as N-terminal6× His tagged proteins. 1 liter cultures were grown at room temperature(˜20° C.) in LB media supplemented with 40 μg/mL kanamycin. Proteinexpression was induced with 500 μM IPTG added at an A₆₀₀ of 1.0. Inducedcultures were allowed to grow for 15 hours (also at room temperature).

Recombinant Δ4, 5 glycuronidase purification. Bacterial cells wereharvested by centrifugation at 6000×g for 20 minutes and resuspended in40 mL of binding buffer (50 mM Tris-HCL, pH 7.9, 0.5 M NaCl, and 10 mMimidazole). Lysis was initiated by the addition of 0.1 mg/mL lysozyme(20 minutes at room temperature) followed by intermittent sonication inan ice-water bath using a Misonex XL sonicator at 40-50% output. Thecrude lysate was fractionated by low-speed centrifugation (18,000×g; 4°C.; 15 minutes) and the supernatant was filtered through a 0.45 micronfilter. The 6×-His tag Δ4, 5 glycuronidase was purified by Ni⁺²chelation chromatography on a 5 mL Hi-Trap column (Pharmacia Biotech,N.J.) pre-charged with 200 mM NiSO₄ and subsequently equilibrated withbinding buffer. The column was run at a flow rate of approximately 3-4mL/minute that included an intermediate wash step with 50 mM imidazole.The Δ4, 5 enzyme was eluted from the column in 5 mL fractions using highimidazole elution buffer (50 mM Tris-HCL, pH 7.9, 0.5 M NaCl, and 250 mMimidazole). Peak fractions were dialyzed overnight against 4 liters ofphosphate buffer (0.1 M sodium phosphate, pH 7.0, 0.5 M NaCl) to removethe imidazole.

The 6× His tag was effectively cleaved by adding biotinylated thrombinat 2 units/milligram of recombinant protein, overnight at 4° C. withgentle inversion. Thrombin was captured by binding to streptavidinagarose at 4° C. for two hours using the Thrombin Capture Kit (Novagen).The cleaved peptide 5′ MGSSHHHHHHSSGLVPR 3′ (SEQ ID NO. 16) was removedby final dialysis against a 1000-fold volume of phosphate buffer.

Protein concentrations were determined by protein assay (Bio-Rad,Hercules, Calif.) and confirmed by UV spectroscopy using a theoreticalmolar extinction coefficient ε=88,900 for Δ4,5²⁰ . Protein purity wasassessed by SDS-PAGE followed by Coomassie Brilliant Blue staining.

Computational methods. Signal sequence predictions were made by SignalPV1.1 using the von Heijne computational method [Nielsen, H.,Engelbrecht, J., Brunak, S., and von Heijne, G., 1997, Protein Eng 10,1-6] with maximum Y and S cutoffs set at 0.36 and 0.88, respectively.Glycuronidase multiple sequence alignments were made from select BLASTPdatabase sequences (with scores exceeding 120 bits and less than 6%gaps) using the CLUSTAL W program (version 1.81) preset to an open gappenalty of 10.0, a gap extension penalty of 0.20, and both hydrophilicand residue-specific gap penalties turned on. Assay for enzyme activityand determination of kinetic parameters. Standard reactions were carriedout at 30° C. and included 100 mM sodium phosphate buffer, pH 6. 4, 50mM NaCl, 500 μM disaccharide substrate and 200 nM enzyme in a 100 μlreaction volume. Hydrolysis of heparin disaccharides was determinedspectrophotometrically by measuring the loss of the Δ4, 5 chromophoremeasured at 232 nm. Substrate hydrolysis was calculated using thefollowing molar extinction coefficients empirically determined for eachdisaccharide substrate: ΔUH_(NAc), ε₂₃₂=4,524; ΔUH_(NAc6S), ε₂₃₂=4,300;ΔUH_(NS), ε₂₃₂=6,600; ΔUH_(NS,6S), ε₂₃₂=6,075; ΔUH_(NH26S), ε₂₃₂=4,826;ΔU_(2S)H_(NS), ε₂₃₂=4,433. Initial rates (V_(o)) were extrapolated fromlinear activities representing <10% substrate turnover and fit to pseudofirst-order kinetics. For kinetic experiments, disaccharideconcentration for each respective substrate was varied from 48 to 400 μMconcentrations. K_(m) and k_(cat) values were extrapolated from V_(o)vs. [S] curves fit to the Michaelis Menten equation by a non-linear,least squares regression.

For experiments measuring the relative effect of ionic strength onglycuronidase activity, the NaCl concentration was varied from 0.05 to 1M in 0.1 M sodium phosphate buffer (pH 6.4), 200 μM ΔUH_(NS,6S) and 100μM enzyme under otherwise standard reaction conditions. The effect of pHon catalytic activity was kinetically determined at varying ΔUH_(NS,6S)concentrations in 0.1 M sodium phosphate buffer at pH 5.2, 5.6, 6.0,6.4, 6.8, 7.2 and 7.8. Data were fit to Michaelis-Menten kinetics asdescribed above and the relative k_(cat)/K_(m) ratios plotted as afunction of pH.

Detection of Δ4, 5 glycuronidase activity by capillary electrophoresis.200 μg of heparin (Celsus Laboratories) was subject to exhaustiveheparinase digestion as described [Venkataraman, G., Shriver, Z., Raman,R., and Sasisekharan, R., 1999, Science 286, 537-42] with certainmodifications that included a 50 mM PIPES buffer, pH 6.5 with 100 mMNaCl in a 100 μl reaction volume. Following heparinase treatment, 25picomoles of Δ4, 5 glycuronidase was added to one-half of the originalreaction (pre-equilibrated at 30° C.). 20 μl aliquots were removed at 1minute and 30 minutes and activity quenched by heating at 95° C. for 10minutes. 20 μl of the minus Δ4, 5 control (also heated for 10 minutes)was used as the 0 time point. Disaccharide products were resolved bycapillary electrophoresis run for 25 minutes under positive polaritymode as previously described [Rhomberg, A. J., Ernst, S., Sasisekharan,R., and Biemann, K., 1998, Proc Natl Acad Sci USA 95, 4176-81].

Molecular mass determinations. Molecular mass determinations werecarried out by MALDI-MS as described [Rhomberg, A. J., Ernst, S.,Sasisekharan, R., and Biemann, K., 1998, Proc Natl Acad Sci USA 95,4176-81].

Example 1 Molecular Cloning of Δ4, 5 Glycuronidase Gene from F.heparinum Genome

To clone the Δ4, 5 glycuronidase gene, we isolated a series of Δ4, 5glycuronidase-derived peptides after protease treatment of the purifiedenzyme. The native enzyme was directly purified from fermentationcultures of F. heparinum using a 5-step chromatography scheme aspreviously described [McLean, M. W., Bruce, J. S., Long, W. F., andWilliamson, F. B., 1984, Eur J Biochem 145, 607-15]. The extent ofpurity was ultimately characterized by reverse phase chromatography,which indicated a single major peak (FIG. 1A). We were able to generatea number of peptides by a limited trypsin digestion of the purifiedenzyme. 26 peptide fragments were resolved by reverse phasechromatography (FIG. 1B). From these 26, at least eight peptides(corresponding to major peaks 8, 12, 13, 19, 24, and 26) were ofsufficient yield and purity and were selected for protein sequencedetermination (FIG. 1C).

Based on this information, we designed degenerate primers correspondingto peaks 19, 24, and 26. These primers were used to PCR amplify Δ4, 5specific sequences to be used as a suitable DNA hybridization probes forscreening the Flavobacterial genomic library. A combination of twoprimer pairs in particular (peak 19 forward and peak 24 reverse) gave adiscrete PCR product of approximately 450 base pairs. The translation ofthe corresponding DNA sequence indicated that it contained the expectedamino acid sequence corresponding to peaks 19 and 24. The peak 12peptide also mapped to this region. We used this discrete PCR amplifiedDNA fragment (designated as probe 1, FIG. 2A) in the initial plaquehybridizations. The most 5′ terminal clone obtained in this screening(represented by clone G5A) included approximately one-half of thepredicted gene size corresponding to the carboxy terminus of theputative ORF. Invariably, all of the isolated clones possessed and EcoR1 site at their respective 5′ termini. In an attempt to isolate a clonefrom the phage library possessing the other half of the gene, werescreened additional plaques, this time using a second N-terminalspecific DNA hybridization probe (probe 2) in tandem with the originalprobe 1. This second strategy also failed to yield any clones with thefully-intact Δ4, 5 gene. A partial, overlapping clone (G5H), however,did extend the known 5′ sequence of A 4, 5 by approximately 540 basepairs. Alternate approaches were taken in an attempt to obtain the 5′terminus of the glycuronidase gene. The size of this missing region wasestimated, based on the molecular weight of the native protein, to beapproximately 45 amino acids (or 135 base pairs). We completed DNAsouthern analyses to identify potentially useful DNA restriction sitesflanking the 5′ end of the Δ4, 5 gene (FIG. 2B). This restrictionmapping ultimately involved the use of the Eco R1 site within the genein conjunction with hybridization probe 3 (whose 3′ end lies just 5′ tothis restriction site) to positively bias our search for the remainingamino terminus. Based on this refined map, we successfully isolated andsubcloned an approximately 1.5 kb Bgl II- Eco R1 Δ4, 5 fragment intopLITMUS. The 5′ terminus of the Δ4, 5 gene was obtained from direct DNAsequencing of this subgenomic clone.

Results

A summary of the full-length gene obtained from the two overlappingcloning methods is depicted in FIG. 2C. The DNA sequence analysiscompiled from the overlapping Δ4, 5 clones (and confirmed by directsequencing of a single PCR amplified genomic clone) is shown in FIG. 3.The Δ4, 5 coding sequence is comprised of 1209 base pairs correspondingto an ORF that encodes a 402 amino acid protein. The amino acid andnucleotide sequences for the full-length enzyme are given as SEQ IDNos.:3 and 4, respectively. The predicted molecular weight of 45,621daltons for the translated protein corresponds very well with anempirical molecular mass of 45,566 daltons for the purified

Flavobacterial enzyme determined by MALDI-MS. All of the peptides forwhich we obtained sequence information map to this Δ4, 5 ORF. Based onfurther primary sequence analyses, we have also identified a likelybacterial signal sequence spanning amino acids 1-20 also possessing aputative cleavage site between residues G20 and M21 (FIG. 4B). Thepresence of a markedly hydrophobic amino terminus (see hydropathy plot,FIG. 4A) and the identification of an AXXA peptidase cleavage motiffurther support this assumption [von Heijne, G., 1988, Biochim BiophysActa 947, 307-33].

A search for related sequences in the NCBI protein database led toseveral functionally related enzymes. In a multiple sequence alignmentof our cloned enzyme with select glucuronyl hydrolases, we found ahomology that generally corresponded to upwards of 30% identity andnearly 50% similarity at the primary sequence level (FIG. 4C). Thisrelatedness spanned most of the enzyme sequence, excluding theN-terminus. Based on this alignment, we found several highly conservedpositions within the F. heparinum Δ4, 5 glycuronidase that included, inparticular, several aromatic and acidic residues. Other invariant aminoacids of possible catalytic importance include H115 and R151.

Example 2 Recombinant Expression and Purification of the Δ4, 5Glycuronidase

Using PCR, we cloned from the F. heparinum genome both the full-lengthenzyme and the “mature” enzyme lacking the N-terminal 20 amino acidsignal sequence)(Δ4,5²⁰ ) into a T7-based expression plasmid. Cloninginto pET28a permitted the expression of the glycuronidase as anN-terminal 6× His-tag fusion protein. Pilot expression studies focusedon the full-length enzyme. In these initial experiments, we examinedseveral different induction conditions such as temperature, time andlength of induction, and even IPTG concentrations. In every case, thefull-length enzyme was present nearly exclusively as an insolublefraction. Attempts to purify the enzyme directly from inclusion bodiesand then refold the apparently mis-folded protein were initially notsuccessful; while solubility was partially achieved by a combined use ofdetergents (e.g., CHAPS), increasing salt concentrations, and thepresence of glycerol, the partially purified enzyme was largelyinactive.

A possible explanation for this insolubility points to the presence of avery hydrophobic region within the wild-type protein sequence spanningthe first 20 amino acids. The N-terminal sequence is also predicted tocomprise a cleavable prokaryotic signal sequence with the most likelycleavage site occurring between position G20 and M21 (FIG. 3). Withinthis sequence, we also find the alanine repeat 5′ (SEQ ID NO. 17) thatmay serve as part of the actual cleavage recognition sequence [vonHeijne, G., 1988, Biochim Biophys Acta 947, 307-33]. This signal peptidewould indicate a periplasmic location for the glycuronidase with theN-terminus of the secreted (mature) protein beginning with M21. Werecombinantly expressed this mature variant)(Δ4,5^(Δ20) ) in which thesignal sequence was replaced entirely by an N-terminal 6× Hispurification tag.

Recombinant expression of the enzyme lacking the presumed signalsequence yielded remarkably different results. In this case, solublerecombinant expression levels routinely reached several hundredmilligrams of protein per liter of induced cells. The specific activityof this enzyme on the heparin disaccharide ΔUH_(Nac) was likewiserobust.

Results

A summary of the expression and purification of Δ4,5²⁰ is summarized inFIG. 5 and Table 1. A two-step purification comprised of Ni⁺² chelationchromatography followed by thrombin cleavage to remove the 6× Hispurification tag, typically yielded over 200 mg of greater than 90% pureenzyme as assessed by SDS-PAGE followed by Coomassie Brilliant Bluestaining. An approximately three-fold purification of activity wasachieved in a single chromatographic step. Most notably, the specificactivity of the recombinant enzyme acting upon ΔUH_(NAc) far exceededthose values reported in the literature [Warnick, C. T. and Linker, A.,1972, Biochemistry 11, 568-72]. While removal of the 6× His tag from theN-terminus of the enzyme was unnecessary for optimal hydrolyticactivity, the presence of the histidine tag did appear to instigateprotein precipitation over an extended time especially at higher enzymeconcentrations. This tag was, therefore removed for all subsequentbiochemical experiments. In this manner, the recombinant protein wasstable at 4° C. for at least two weeks during which time it remained insolution at protein concentrations exceeding 10 mg/mL under the bufferconditions described.

A molecular mass of 44,209 daltons was determined for the recombinantenzyme (i.e., Δ4,5²⁰ ) by MALDI-MS. The amino acid and nucleotidesequence for the enzyme which lacks the N-terminal 20 amino acid signalsequence is given as SEQ ID Nos.:1 and 2, respectively. Thisempirically-established molecular weight is consistent with itstheoretical value of 43,956 Daltons based on its amino acid composition.This value physically differs by 1357 Daltons in comparison to amolecular weight of 45,566 daltons likewise measured for the nativeenzyme. This mass differential is largely accounted for by theengineered removal in the recombinant protein of the 20 amino acidsignal sequence. However, we cannot exclude the possibility ofdifferential posttranslational modifications such as glycosylationlargely accounting for the observed differences between the two enzymepopulations. Unfortunately, chemical blocking precluded us fromdetermining the N-terminal sequence of the native protein.

TABLE 1 Purification summary for the recombinant Δ4, 5 glycuronidase.Sp. Activity Protein (μmoles DiS/min./ % Purification Step Yield (mg) mgprotein) purification Crude lysate 400 4.7 — Ni⁺² chromatography 20512.9 2.7 Thrombin cleavage 205 13.6 2.9 Specific activities for eachfraction were measured using 800 ng of protein and 120 μM of theunsulfated heparin disaccharide (DiS) ΔUH_(NAc) in a 100 μl reactionvolume. The fold purification was calculated relative to the specificactivity measured for the crude lysate.

Example 3 Biochemical Conditions for Optimal Enzyme Activity

To determine the optimal reaction conditions for Δ4, 5 glycuronidaseactivity, we analyzed initial reaction rates as a function of buffer,pH, temperature, and ionic strength (FIG. 6). For these experiments, weused the disulfated heparin disaccharide substrate ΔUH_(NS,6S). Based onwhat is known about the degradation of heparin/heparan sulfate-likeglycosaminoglycans by flavobacteria as well as initial biochemicalcharacterization of this and related enzymes [Warnick, C. T. and Linker,A., 1972, Biochemistry 11, 568-72], we hypothesized that a heparindisaccharide would be an optimal substrate for the Δ4, 5 glycuronidase.Enzyme activity was routinely monitored by a loss of absorbance at 232nm, corresponding indirectly to the hydrolysis of the uronic acid fromthe non-reducing end.

Results

Under these conditions, we observed a NaCl concentration-activitydependence that was optimal between 50 and 100 mM. NaCl concentrationsexceeding 100 mM demonstrated a significant and relatively sharplynegative effect on specific activity (FIG. 6A), i.e., with approximately60% inhibition occurring at 250 mM NaCl relative to 100% activitymeasured at 100 mM NaCl. The steep transition observed in the NaCltitration curve suggests an important role of ionic interactions in someaspect of enzymatic function.

The observed pH profile for the glycuronidase is bell-shaped (FIG. 6C)with a pH optimum of 6.4. Interestingly, initial reaction rates aresignificantly reduced at the highest temperatures measured, especiallyat 42° C. (FIG. 6B). Precincubation experiments at 30, 37, and 42° C. toassess relative enzyme stabilities at these temperatures, however,indicated no significant change in relative enzyme activities whensubsequently measured under the standard 30° C. reaction conditions. Theresults of such an experiment strongly suggest that thermal lability isnot the issue.

As a final variable for optimizing Δ4, 5 glycuronidase in vitro reactionconditions, we also considered any requirement for divalent metal ions.We found no evidence that metals are either required for catalysis oractivate the enzyme to any appreciable extent.

Having established the reaction conditions for optimal Δ4, 5glycuronidase activity, we next compared the specific activity of therecombinant enzyme)(Δ4,5^(Δ20) ) relative to the native enzyme purifieddirectly from F. heparinum (FIG. 7). The activities of both enzymefractions were measured in parallel under identical reaction conditions.In this comparison, the recombinant Δ4, 5 possessed an approximatelythree-fold higher specific glycuronidase activity relative to the nativeenzyme. These observed rates demonstrate quite clearly that the clonedΔ4, 5 enzyme possesses “wild-type” activity that is in no way adverselyaffected by its recombinant expression in E. coli.

Example 4 Δ4, 5 Glycuronidase Substrate Specificity

The specificity of the Δ4, 5 glycuronidase acting on variousglycosaminoglycan disaccharide substrates was investigated. The varioussubstrates examined included both heparin and chondroitin disaccharidesas well as an hyaluronadate. In particular, we considered thepossibility of any structural discriminations pertaining to glycosidiclinkage position (1→4 vs. 1→3) and relative sulfation state within thedisaccharide. For each substrate, kinetic parameters were determinedbased on substrate saturation profiles that fit Michaelis-Mentenassumptions (FIG. 8). These kinetic values are listed in Table 2. Forthe heparin disaccharides, k_(cat) values varied significantly fromapproximately 2 to 15 sec⁻¹, while the apparent K_(m) values for eachrespective disaccharide were comparable, ranging from approximately100-300 μM.

Results

The heparin disaccharide ΔU_(2S)H_(NS) was not a substrate at any of theconcentrations studied, even following an extended incubation timespanning several hours. For those heparin disaccharides that werehydrolyzed under the conditions tested and for which kinetic parameterscould be determined, an interesting substrate preference was apparent.In this hierarchy and under these conditions, the two disaccharidesΔUH_(NAc) and ΔUH_(Nac6S) were the best substrates; in comparison,ΔUH_(NH26S) and ΔUH_(NS) were less good as substrates. The kineticvalues for ΔUH_(NS,6S) fell roughly in the middle between these twoboundaries.

The data show that heparin is a better substrate thanchondroitin/dermatan and/or hyaluronan, although these compounds arealso substrates. None of the non-heparin disaccharides were hydrolyzedunder the conditions for measuring substrate kinetics. This resultindicates an unequivocal discrimination of the Δ4, 5 in regard tolinkage position, with a strong preference for 1→4 versus 1→3 linkages.At the same time, these disaccharides were slowly hydrolyzed to varyingdegrees when the enzyme reactions were conducted over a much longertimecourse (>12 hours) and at considerably higher enzyme concentrations.After approximately 18 hours, greater than 80% of monosulfatedchondroitin disaccharide (ΔUGal_(Nac6S)) was hydrolyzed, whereas thenon-sulfated chondroitin (ΔUGal_(NAc)) and the hyaluronan disaccharide(ΔUH) were still present at approximately 40% and 65%, respectively. Theimportance of the linkage position is, therefore, not absolute. Theapparently positive effect of chondroitin 6-O-sulfation within thegalactosamine is consistent with our results for the heparin substratesand provides further evidence for the influence of this position indictating substrate specificity.

Based on the kinetically defined substrate specificity for thedisaccharides, we set out to validate these results while, at the sametime, to investigate the utility of the Δ4, 5 glycuronidase as anenzymatic tool for HSGAG compositional analyses. In this manner, the Δ4,5 should be very useful in assessing the composition of disaccharidesresulting from prior heparinase treatment of heparin/heparan sulfate.For this particular experiment, we pre-treated 200 μg of heparin with aheparinase cocktail. This exhaustive digestion was then directlyfollowed by a relatively short (1 minute) or long (30 minute) Δ4, 5glycuronidase treatment carried out under optimal reaction conditions.The disaccharide products were then resolved by capillaryelectrophoresis. The electrophoretic mobility profile for the Δ4, 5treated saccharides were then directly compared to the untreated control(i.e., heparinase treatment only) run under identical conditions (FIG.9). 7 disaccharide peaks were present in the capillaryelectrophoretogram corresponding to the heparinase only control (A.). Astructural assignment for each one of these peaks was made based onpreviously established compositional analyses. For the most part, theresolution of these Δ4, 5 containing saccharides was such that eachalternating peak (1, 3, 5, . . . ) corresponded to a disaccharidepossessing a 2-O sulfated uronic acid at the non-reducing end.Predictably, the relative amplitude and area of these peaks remained thesame, over the entire timecourse of the Δ4, 5 preincubation. Thisunchanging result extended to 18 hours. On the other hand, peakscorresponding to disaccharides lacking the 2-O sulfate were eliminated.Moreover, the relative rates of their disappearances elegantlycorresponded to their respective preferences as substrates as determinedin the previous kinetic experiment. ΔUH_(NAc6S) (peak 8) was essentiallyhydrolyzed within one minute; ΔUH_(NS,6S) (peak 4) and ΔUH_(NS) (peak 6)were approximately 75% and 50% hydrolyzed, respectively. These twolatter substrates were completely depleted by 30 minutes.

In addition to the assigned disaccharides, the Δ4, 5 glycuronidase alsoacted on a heparinase-generated tetrasaccharide (identified as peak 2 inFIG. 9). The assignment of Peak 2 as a tetrasaccharide was confirmed byMALDI-MS indicating a mass of 1036.9 that corresponds to a singlyacetylated tetrasaccharide with four sulfates. Disaccharide analysis ofthis tetrasaccharide further indicated that it does not contain a 2-Osulfate at the non-reducing end. The Δ4, 5 enzyme hydrolyzedapproximately one-half of the starting material after one minute. Therelative rate of hydrolysis for this tetrasaccharide roughlycorresponded to the rate observed for the disaccharide ΔUH_(NS) (peak6). This exciting result clearly indicates that a longer chainsaccharide such as a tetrasaccharide is in fact a substrate for the Δ4,5 catalyzed hydrolysis.

TABLE 2 Kinetic parameters for heparin disaccharides. k_(cat) K_(m)Relative Disaccharide substrates (sec⁻¹) (μM) k_(cat)/K_(m)k_(cat)/K_(m) ΔUH_(Nac) 15.3 ± 0.9  283 ± 31 0.054 0.49 ΔUH_(NAc, 6S)11.7 ± 0.6  107 ± 15 0.110 1.0  ΔUH_(NS) 4.9 ± 0.4 251 ± 40 0.020 0.18ΔUH_(NS, 6S) 8.8 ± 0.9 334 ± 57 0.026 0.24 ΔUH_(NH2, 6S) 2.4 ± 0.2 235 ±40 0.010 0.09 ΔU_(2S)H_(NS) N.A. N.A. N.A. N.A. k_(cat) and K_(m) valueswere determined from non-linear regression analyses of theMichaelis-Menten curves presented in FIG. 9. * N.A., no activity wasdetected forΔU_(2S)H_(NS).

Each of the foregoing patents, patent applications and references thatare recited in this application are herein incorporated in theirentirety by reference. Having described the presently preferredembodiments, and in accordance with the present invention, it isbelieved that other modifications, variations and changes will besuggested to those skilled in the art in view of the teachings set forthherein. It is, therefore, to be understood that all such variations,modifications, and changes are believed to fall within the scope of thepresent invention as defined by the appended claims.

We claim: 1-19. (canceled)
 20. A method of cleaving a glycosaminoglycan,comprising: contacting a glycosaminoglycan with a Δ4,5 unsaturatedglycuronidase in an effective amount to cleave the glycosaminoglycan.21-39. (canceled)
 40. A method of inhibiting angiogenesis, comprisingadministering to a subject in need thereof an effective amount of apharmaceutical preparation comprising a Δ4,5 unsaturated glycuronidaseor an expression vector encoding the glycuronidase.
 41. The method ofclaim 40, wherein the subject is administered with the pharmaceuticalpreparation in an amount effective in treating cancer.
 42. The method ofclaim 40, wherein the subject is administered with the pharmaceuticalpreparation in an amount effective in inhibiting cellular proliferation.43. A method of treating a coagulation disorder, comprisingadministering to a subject in need thereof an effective amount of apharmaceutical preparation comprising a Δ4,5 unsaturated glycuronidaseor an expression vector encoding the glycuronidase. 44-49. (canceled)